1,5-disubstituted 1,2,3-triazoles are inhibitors of RAC/CDC42 GTPases

ABSTRACT

Compounds are disclosed that inhibit RhoGTPases that are useful for inhibiting hyperprofilerative and neoplastic diseases. Specifically, the compounds inhibit the GTPases Rac and Cdc42 that are overactive or overexpressed in signaling pathways in cancer and metastasis. Methods for treatment of cancer and hyperproliferative diseases are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of copending U.S. patent applicationSer. No. 15/499,532, filed Apr. 27, 2017, which claims priority fromU.S. Ser. No. 62/328,282, filed Apr. 26, 2016. The disclosures set forthin the referenced applications are incorporated herein by reference intheir entireties.

This invention was made with Government support under Award Nos.NIH/NIHMD P20GM103475; NIH/NIGMS SC3GM116713; NIH/NIMHD G12MD007600 andNIH/NCI U54 CA096297 awarded by The National Institutes of Health. TheU.S. Government has certain rights in the invention.

BACKGROUND

Compounds are disclosed that inhibit Rho GTPases that are useful forinhibiting hyperprofilerative and neoplastic diseases. Specifically, thecompounds inhibit the GTPases Rac and Cdc42 that are overactive oroverexpressed in signaling pathways in cancer and metastasis. Methodsfor treatment of cancer and hyperproliferative diseases are disclosed.

The Rho GTPases Rac (Ras-related C3 botulinum toxin substrate) and Cdc42(cell division control protein 42 homolog) regulate cell functionsgoverning cancer malignancy, including cell polarity, migration, andcell cycle progression. The Rho family of GTPases in humans consists of20 different members, and aberrant behavior in their regulatory activityhas been implicated in cancer and other diseases. More than 70 Guaninenucleotide Exchange Factors (GEFs) are known, which specificallyactivate one or more of the GTPases. In turn, the activated GTPases canspecifically interact with over 60 downstream effectors. Dysregulationof one or more cellular processes can lead to release of malignant cellsfrom their original locations, which subsequently can establishthemselves in pre-metastatic niches in, for example, bone or lungs. Ithas been found that members of the Rho GTPase family, including Rac,Cdc42 and Rho, play key signaling roles in these processes.

Rho GTPases regulate migration and invasion, cytoskeletal organization,transcriptional regulation, cell cycle progression, apoptosis, vesicletrafficking, and cell-to-cell and cell-to-extracellular matrixadhesions. The Rho GTPases Rac and Cdc42 are potent inducers of actinpolymerization and extension of actin structures at the leading edge ofmotile cells. In addition, Cdc42 plays a critical role in cell polarity,and thus, promotes directed and persistent migration.

Studies have implicated hyperactive Rac and Cdc42 with increased cancercell survival, proliferation, and invasion, as well in Ras and otheroncogene-mediated transformation. Furthermore, oncogenic cell surfacereceptors, such as tyrosine kinase, cytokine, and G protein coupledreceptors, activate Rac and Cdc42 via regulation of their upstreameffector GEFs. Accordingly, Rac and Cdc42 proteins are generally notmutated in cancer but rather overexpressed or hyperactivated. Eventhough ˜9% of melanomas contain an activating Rac(P29S) mutation, andthe hyperactive splice variant Rac1b is overexpressed in some cancers, amajority of the Rac and Cdc42 in human cancer are activated due toupregulated GEFs.

Of the direct downstream effectors of Rac and Cdc42, p21-activatedkinases (PAK) are overexpressed in a number of cancers and contribute tocancer transformation and progression by regulating key cellularfunctions, including cytoskeletal organization, cell migration,adhesion, growth, and development. Therefore, a number of PAK inhibitorshave been developed as anti-cancer therapeutics. However, these havebeen limited by specificity, bioavailability, and toxicity, and have yetto successfully complete clinical trials.

There is a need for new therapeutic agents for the treatment of cancerand other hyperproliferative diseases. The Rac and Cdc42 GTPases areimportant cellular mediators that are hyperactive or overexpressed inmetastatic tumors. Design of novel inhibitors of the activities of Racand/or Cdc42 with improved activity, pharmacochemical profile andreduced toxicity is desirable.

SUMMARY

A series of novel 1,5-disubstituted 1,2,3-triazoles is disclosed herein.The inventors developed Rac inhibitor EHop-016 (IC₅₀, 1,100 nM) thatinhibits cancer cell migration and viability, and reduces tumor growth,metastasis, and angiogenesis in vivo. Compound EHop-016 was reported toinhibit Rac1 activity at concentrations <5 μM and with an IC50=1.1 μM inMDA-MB-435 metastatic cells. At higher concentrations (>10 μM) EHop-016inhibits Rac activity by 100% and Cdc42 activity by 75%. Ehop-016inhibited in vitro cell migration, and in an in vivo model formetastatic cancer in mice was able to inhibit metastasis and tumorgrowth.

Some of the members are approximately 10 times more potent inhibitors ofRac and Cdc42 than EHop-016. Treatment of MDA-MB-435 cells with 150 nMof a specific example from this series (MBQ-167) for 24 h resulted inreduced expression of oncogenes and survival inducers c-Myc, Bcl-XL,Bcl-2, with a concomitant increase in the pro-apoptotic protein BAD.Studies from a number of cancer types have shown that Rac/Cdc42/PAKsignaling can induce cell survival and evade apoptosis. Therefore, thedecreased cell viability and increased apoptosis by MBQ-167, atconcentrations that inhibit both Rac and Cdc42, is predicted to be dueto dual inhibition of Rac and Cdc42 function.

MBQ-167 inhibits Rac and Cdc42 with IC₅₀s of 103 nM and 78 nMrespectively, in metastatic breast cancer cells. Consequently, MBQ-167significantly decreases Rac and Cdc42 downstream effector p21-activatedkinase (PAK) signaling and the activity of signal transducer andactivator of transcription (STAT3), without affecting Rho, MAPK, or Aktactivities. MBQ-167 also inhibits breast cancer cell migration,viability, and mammosphere formation. Moreover, MB Q-167 affects cancercells that have undergone epithelial to mesenchymal transition by a lossof cell polarity, and inhibition of cell surface actin-based extensions,to ultimately result in detachment from the substratum. Prolongedincubation (120 h) in MBQ-167 decreases metastatic cancer cell viabilitywith a GI₅₀ of ˜130 nM, without affecting non-cancer mammary epithelialcells. The loss in cancer cell viability is due to MBQ-167-mediated G₂/Mcell cycle arrest and subsequent apoptosis, especially of the detachedcells. In vivo, MBQ-167 inhibits mammary tumor growth and metastasis inimmunocompromised mice by ˜90%. In conclusion, MBQ-167 is 10× morepotent than other currently available Rac/Cdc42 inhibitors, and haspotential to be developed as an anticancer drug, as well as a dualinhibitory probe for the study of Rac and Cdc42.

The following numbered embodiments are contemplated and arenon-limiting:

1. A compound of the formula (I),

-   -   wherein A and B are independently H, deuterium, halogen, C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₆-C₁₀        aryl, —OH, —CN, —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂,        —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)C₁-C₆ alkyl, —OR⁴, CO₂H,        —C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆        alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl,        —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl); or A and B taken        together with the ring to which they are attached form a C₅-C₈        cycloalkyl, C₆-C₁₀ aryl, or a 5- to 8-membered heterocycloalkyl;        wherein each hydrogen atom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, C₃-C₆ cycloalkyl, C₅-C₈ cycloalkyl, C₆-C₁₀ aryl, or 5-        to 8-membered heterocycloalkyl is independently optionally        substituted by deuterium, halogen, —OH, —CN, —OR⁴, —OC₁-C₆        alkyl, —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆        alkyl, —N(C₁-C₆ alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂,        —NHC(O)NHC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)NH₂, —N(C₁-C₆        alkyl)C(O)NHC₁-C₆ alkyl, —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆        alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆        alkyl)C(O)OC₁-C₆ alkyl, —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆        alkyl), —N(C₁-C₆ alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆        alkyl)S(O)₂(C₁-C₆ alkyl), —NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆        alkyl)S(O)NH₂, —N(C₁-C₆ alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl),        —NHS(O)₂NH(C₁-C₆ alkyl), —NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆        alkyl)₂, —N(C₁-C₆ alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆        alkyl)S(O)₂NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)N(C₁-C₆ alkyl)₂,        —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆ alkyl)₂, —CO₂H, —C(O)OC₁-C₆ alkyl,        —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆        alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆        alkyl), —S(O)₂NH(C₁-C₆ alkyl), —S(O)N(C₁-C₆ alkyl)₂,        —S(O)₂N(C₁-C₆ alkyl)₂, —P(C₁-C₆ alkyl)₂, —P(O)(C₁-C₆ alkyl)₂,        —C₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl, —CF₃,        —CHF₂, or —CH₂F;    -   R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆        cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, or        mono- or bicyclic heteroaryl; wherein each hydrogen atom in        C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3-        to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, or mono- or        bicyclic heteroaryl is independently optionally substituted by        deuterium, halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or —OR⁵;    -   each R² is independently deuterium, halogen, —OH, —CN, C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₆-C₁₀        aryl, —OR⁶, —C(O)OR⁶, —C(O)NR⁶R⁷, —NH₂, —NH(C₁-C₆ alkyl),        —N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —CO₂H, —C(O)OC₁-C₆ alkyl,        —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆        alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆        alkyl), —S(O)₂NH(C₁-C₆ alkyl); wherein each hydrogen atom in        C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, and        C₆-C₁₀ aryl is independently optionally substituted by        deuterium, halogen, —OH, —CN, —OR⁶, —OC₁-C₆ alkyl, —NH₂,        —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆        alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl,        —N(C₁-C₆ alkyl)C(O)NH₂, —N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl,        —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂,        —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)OC₁-C₆ alkyl,        —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl), —N(C₁-C₆        alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),        —NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆        alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),        —NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆        alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl),        —N(C₁-C₆ alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆        alkyl)₂, —CO₂H, —C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆        alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl,        —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl),        —S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆ alkyl)₂, —P(C₁-C₆ alkyl)₂,        —P(O)(C₁-C₆ alkyl)₂, —C₃-C₆ cycloalkyl, or 3- to 7-membered        heterocycloalkyl;    -   R³ is independently H, deuterium, C₁-C₆ alkyl, C₂-C₆ alkenyl,        C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to 7-membered        heterocycloalkyl, C₆-C₁₀ aryl, C₆-C₁₀ aryl-(C₆-C₁₀ aryl), C₁-C₆        alkyl-(NHR⁸), C₁-C₆ alkyl-(OR⁸), C₆-C₁₀ aryl-(OR⁸), C₁-C₆        alkyl-(NR⁸R⁹), or mono- or bicyclic heteroaryl; wherein each        hydrogen atom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        C₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C₁₀        aryl, or mono- or bicyclic heteroaryl is optionally substituted        by deuterium, halogen, —OH, oxo, —OR⁸, —NHR⁸, —CN, —OC₁-C₆        alkyl, —NH₂, —NH(C₁-C₆ alkyl), —NH(C₆-C₁₀ aryl), —NH(C₆-C₁₀        aryl)-N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆        alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl,        —N(C₁-C₆ alkyl)C(O)NH₂, —N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl,        —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂,        —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)OC₁-C₆ alkyl,        —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl), —N(C₁-C₆        alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),        —NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆        alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),        —NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆        alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl),        —N(C₁-C₆ alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆        alkyl)₂, —CO₂H, —C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆        alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl,        —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl),        —S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆ alkyl)₂, —P(C₁-C₆ alkyl)₂,        —P(O)(C₁-C₆ alkyl)₂, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆        cycloalkyl, 3- to 7-membered heterocycloalkyl, C₁-C₆ alkyl-(3-        to 7-membered heterocycloalkyl), —CF₃, —CHF₂, or —CH₂F;    -   each R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ is independently H, deuterium,        C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3-        to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, C₁-C₆ alkyl-(3- to        7-membered heterocycloalkyl), heteroaryl, —S(O)₂C₁-C₆ alkyl,        —CF₃, —CHF₂, or —CH₂F, wherein each hydrogen in C₆-C₁₀ aryl is        optionally substituted by C₃-C₆ cycloalkyl, 3- to 7-membered        heterocycloalkyl, or —CH₂CN; and    -   x is 0, 1, 2, or 3;    -   or a pharmaceutically acceptable salt thereof.

2. The compound of clause 1, wherein A and B taken together with thering to which they are attached form a C₅-C₈ cycloalkyl, C₆-C₁₀ aryl, ora 5- to 8-membered heterocycloalkyl, wherein each hydrogen atom in C₅-C₈cycloalkyl, C₆-C₁₀ aryl, or a 5- to 8-membered heterocycloalkyl isindependently optionally substituted by deuterium, halogen, —OH, —CN,—OR⁴, —OC₁-C₆ alkyl, —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂,—NHC(O)C₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂,—NHC(O)NHC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)NH₂, —N(C₁-C₆ alkyl)C(O)NHC₁-C₆alkyl, —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂,—NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)OC₁-C₆ alkyl, —NHS(O)(C₁-C₆alkyl), —NHS(O)₂(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl), —NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆alkyl)S(O)NH₂, —N(C₁-C₆ alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl),—NHS(O)₂NH(C₁-C₆ alkyl), —NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆alkyl)₂, —N(C₁-C₆ alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)S(O)₂NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)S(O)₂N(C₁-C₆ alkyl)₂, —CO₂H, —C(O)OC₁-C₆ alkyl, —C(O)NH₂,—C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl),—S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆ alkyl)₂, —P(C₁-C₆ alkyl)₂,—P(O)(C₁-C₆ alkyl)₂, —C₃-C₆ cycloalkyl, 3- to 7-memberedheterocycloalkyl, —CF₃, —CHF₂, or —CH₂F, or a pharmaceuticallyacceptable salt thereof.

3. The compound of clause 1 or clause 2, wherein A and B taken togetherwith the ring to which they are attached form a C₆-C₁₀ aryl, whereineach hydrogen atom in C₆-C₁₀aryl is independently optionally substitutedby deuterium, halogen, —OH, —CN, —OR⁴, —OC₁-C₆ alkyl, —NH₂, —NH(C₁-C₆alkyl), —N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)C₁-C₆alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)NH₂,—N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl, —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆alkyl)C(O)OC₁-C₆ alkyl, —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),—NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),—NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆ alkyl)₂, —CO₂H,—C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂,—SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆alkyl), —S(O)₂NH(C₁-C₆ alkyl), —S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆alkyl)₂, —P(C₁-C₆ alkyl)₂, —P(O)(C₁-C₆ alkyl)₂, —C₃-C₆ cycloalkyl, 3- to7-membered heterocycloalkyl, —CF₃, —CHF₂, or —CH₂F, or apharmaceutically acceptable salt thereof.

4. The compound of any one of clauses 1-3, wherein R¹ is H, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, or 3- to 7-memberedheterocycloalkyl, wherein each hydrogen atom in C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, or 3- to 7-memberedheterocycloalkyl is independently optionally substituted by deuterium,halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or —OR⁵, or a pharmaceuticallyacceptable salt thereof.

5. The compound of any one of clauses 1-4, wherein R¹ is H, C₁-C₆ alkyl,C₃-C₆ cycloalkyl, or 3- to 7-membered heterocycloalkyl, wherein eachhydrogen atom in C₁-C₆ alkyl, C₃-C₆ cycloalkyl, or 3- to 7-memberedheterocycloalkyl is independently optionally substituted by deuterium,halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or —OR⁵, or a pharmaceuticallyacceptable salt thereof.

6. The compound of any one of clauses 1-5, wherein R¹ is H or C₁-C₆alkyl, wherein each hydrogen atom in C₁-C₆ alkyl is independentlyoptionally substituted by deuterium, halogen, C₁-C₆ alkyl, C₁-C₆haloalkyl, or —OR⁵.

7. The compound of any one of clauses 1-6, wherein x is 0, or apharmaceutically acceptable salt thereof.

8. The compound of any one of clauses 1-6, wherein x is 1, 2, or 3, or apharmaceutically acceptable salt thereof.

9. The compound of any one of clauses 1-8, wherein R³ is C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to 7-memberedheterocycloalkyl, C₆-C₁₀ aryl, C₆-C₁₀ aryl-(C₆-C₁₀ aryl), C₁-C₆alkyl-(NHR⁸), C₁-C₆ alkyl-(OR⁸), C₆-C₁₀ aryl-(OR⁸), C₁-C₆ alkyl-(NR⁸R⁹),or mono- or bicyclic heteroaryl; wherein each hydrogen atom in C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to 7-memberedheterocycloalkyl, C₆-C₁₀ aryl, or mono- or bicyclic heteroaryl isoptionally substituted by deuterium, halogen, —OH, oxo, —OR⁸, —NHR⁸,—CN, —OC₁-C₆ alkyl, —NH₂, —NH(C₁-C₆ alkyl), —NH(C₆-C₁₀ aryl), —NH(C₆-C₁₀aryl)-N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)C₁-C₆alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)NH₂,—N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl, —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆alkyl)C(O)OC₁-C₆ alkyl, —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),—NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),—NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆ alkyl)₂, —CO₂H,—C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂,—SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆alkyl), —S(O)₂NH(C₁-C₆ alkyl), —S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆alkyl)₂, —P(C₁-C₆ alkyl)₂, —P(O)(C₁-C₆ alkyl)₂, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, —C₃-C₆ cycloalkyl, 3- to 7-memberedheterocycloalkyl, C₁-C₆ alkyl-(3- to 7-membered heterocycloalkyl), —CF₃,—CHF₂, or —CH₂F, or a pharmaceutically acceptable salt thereof.

10. The compound of any one of clauses 1-9, wherein R³ is C₁-C₆ alkyl,C₂-C₆ alkenyl, C₆-C₁₀ aryl, C₁-C₆ alkyl-(OR⁸), C₆-C₁₀ aryl-(OR⁸), ormonocyclic heteroaryl; wherein each hydrogen atom in C₁-C₆ alkyl, C₂-C₆alkenyl, C₆-C₁₀ aryl, or monocyclic heteroaryl is optionally substitutedby deuterium, halogen, —OH, oxo, —OR⁸, —NHR⁸, —CN, —OC₁-C₆ alkyl, —NH₂,—NH(C₁-C₆ alkyl), —NH(C₆-C₁₀ aryl), —NH(C₆-C₁₀ aryl)-N(C₁-C₆ alkyl)₂,—NHC(O)C₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂,—NHC(O)NHC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)NH₂, —N(C₁-C₆ alkyl)C(O)NHC₁-C₆alkyl, —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂,—NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)OC₁-C₆ alkyl, —NHS(O)(C₁-C₆alkyl), —NHS(O)₂(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl), —NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆alkyl)S(O)NH₂, —N(C₁-C₆ alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl),—NHS(O)₂NH(C₁-C₆ alkyl), —NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆alkyl)₂, —N(C₁-C₆ alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)S(O)₂NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)S(O)₂N(C₁-C₆ alkyl)₂, —CO₂H, —C(O)OC₁-C₆ alkyl, —C(O)NH₂,—C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl),—S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆ alkyl)₂, —P(C₁-C₆ alkyl)₂,—P(O)(C₁-C₆ alkyl)₂, C₁-C₆ alkyl, C₂- C₆ alkenyl, C₂-C₆ alkynyl, —C₃-C₆cycloalkyl, 3- to 7-membered heterocycloalkyl, C₁-C₆ alkyl-(3- to7-membered heterocycloalkyl), —CF₃, —CHF₂, or —CH₂F, or apharmaceutically acceptable salt thereof.

11. The compound of any one of clauses 1-9, wherein R³ is C₁-C₆ alkyl,C₂-C₆ alkenyl, C₆-C₁₀ aryl, C₁-C₆ alkyl-(OR⁸), C₆-C₁₀ aryl-(OR⁸), ormonocyclic heteroaryl; wherein each hydrogen atom in C₁-C₆ alkyl, C₂-C₆alkenyl, C₆-C₁₀ aryl, or monocyclic heteroaryl is optionally substitutedby deuterium, halogen, —OH, —OR⁸, —OC₁-C₆ alkyl, C₁-C₆ alkyl, or 3- to7-membered heterocycloalkyl, or a pharmaceutically acceptable saltthereof.

12. The compound of clause 1, wherein the compound is selected from thegroup consisting of

or a pharmaceutically acceptable salt thereof.

13. The compound of clause 1 or clause 12, wherein the compound isselected from the group consisting of

or a pharmaceutically acceptable salt thereof.

14. The compound of clause 1, wherein the compound has the formula (II),

-   -   wherein R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        C₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C₁₀        aryl, or mono- or bicyclic heteroaryl; wherein each hydrogen        atom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆        cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, or        mono- or bicyclic heteroaryl is independently optionally        substituted by deuterium, halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl,        or —OR⁵;    -   each R² is independently deuterium, halogen, —OH, —CN, C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₆-C₁₀        aryl, —OR⁶, —C(O)OR⁶, —C(O)NR⁶R⁷, —NH₂, —NH(C₁-C₆ alkyl),        —N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —CO₂H, —C(O)OC₁-C₆ alkyl,        —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆        alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆        alkyl), —S(O)₂NH(C₁-C₆ alkyl); wherein each hydrogen atom in        C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl and        C₆-C₁₀ aryl is independently optionally substituted by        deuterium, halogen, —OH, —CN, —OR⁶, —OC₁-C₆ alkyl, —NH₂,        —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆        alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl,        —N(C₁-C₆ alkyl)C(O)NH₂, —N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl,        —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂,        —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)OC₁-C₆ alkyl,        —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl), —N(C₁-C₆        alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),        —NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆        alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),        —NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆        alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl),        —N(C₁-C₆ alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆        alkyl)₂, —CO₂H, —C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆        alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl,        —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl),        —S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆ alkyl)₂, —P(C₁-C₆ alkyl)₂,        —P(O)(C₁-C₆ alkyl)₂, —C₃-C₆ cycloalkyl, or 3- to 7-membered        heterocycloalkyl;    -   R³ is independently H, deuterium, C₁-C₆ alkyl, C₂-C₆ alkenyl,        C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to 7-membered        heterocycloalkyl, C₆-C₁₀ aryl, C₆-C₁₀ aryl-(C₆-C₁₀ aryl), C₁-C₆        alkyl-(NHR⁸), C₁-C₆ alkyl-(OR⁸), C₆-C₁₀ aryl-(OR⁸), C₁-C₆        alkyl-(NR⁸R⁹), or mono- or bicyclic heteroaryl; wherein each        hydrogen atom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        C₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C₁₀        aryl, or mono- or bicyclic heteroaryl is optionally substituted        by deuterium, halogen, —OH, oxo, —OR⁸, —NHR⁸, —CN, —OC₁-C₆        alkyl, —NH₂, —NH(C₁-C₆ alkyl), —NH(C₆-C₁₀ aryl), —NH(C₆-C₁₀        aryl)-N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆        alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl,        —N(C₁-C₆ alkyl)C(O)NH₂, —N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl,        —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂,        —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)OC₁-C₆ alkyl,        —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl), —N(C₁-C₆        alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),        —NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆        alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),        —NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆        alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl),        —N(C₁-C₆ alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆        alkyl)₂, —CO₂H, —C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆        alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl,        —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl),        —S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆ alkyl)₂, —P(C₁-C₆ alkyl)₂,        —P(O)(C₁-C₆ alkyl)₂, C₂-C₆ alkenyl, C₂-C₆ alkynyl, —C₃-C₆        cycloalkyl, 3- to 7-membered heterocycloalkyl, C₁-C₆ alkyl-(3-        to 7-membered heterocycloalkyl), —CF₃, —CHF₂, or —CH₂F;    -   each R⁵, R⁶, R⁷, R⁸, and R⁹ is independently H, deuterium, C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to        7-membered heterocycloalkyl, C₆-C₁₀ aryl, C₁-C₆ alkyl-(3- to        7-membered heterocycloalkyl), heteroaryl, —S(O)₂C₁-C₆ alkyl,        —CF₃, —CHF₂, or —CH₂F, wherein each hydrogen in C₆-C₁₀ aryl is        optionally substituted by C₃-C₆ cycloalkyl, 3- to 7-membered        heterocycloalkyl, or —CH₂CN; and    -   x is 0, 1, 2, or 3;    -   or a pharmaceutically acceptable salt thereof. The compound of        clause of 1 or clause 14, wherein R1 is H, C1-C6 alkyl, C2-C6        alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, or 3- to 7-membered        heterocycloalkyl, wherein each hydrogen atom in C1-C6 alkyl,        C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, or 3- to        7-membered heterocycloalkyl is independently optionally        substituted by deuterium, halogen, C1-C6 alkyl, C1-C6 haloalkyl,        or —OR5, or a pharmaceutically acceptable salt thereof.

15. The compound of clause of 1 or clause 14, wherein R¹ is H, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, or 3- to7-membered heterocycloalkyl, wherein each hydrogen atom in C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, or 3- to 7-memberedheterocycloalkyl is independently optionally substituted by deuterium,halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or —OR⁵, or a pharmaceuticallyacceptable salt thereof.

16. The compound of any one of clauses 1, clause 14, or clause 15,wherein R¹ is H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, or 3- to 7-memberedheterocycloalkyl, wherein each hydrogen atom in C₁-C₆ alkyl, C₃-C₆cycloalkyl, or 3- to 7-membered heterocycloalkyl is independentlyoptionally substituted by deuterium, halogen, C₁-C₆ alkyl, C₁-C₆haloalkyl, or —OR⁵, or a pharmaceutically acceptable salt thereof.

17. The compound of any one of clauses 1 or clauses 14-16, wherein R¹ isH or C₁-C₆ alkyl, wherein each hydrogen atom in C₁-C₆ alkyl isindependently optionally substituted by deuterium, halogen, C₁-C₆ alkyl,C₁-C₆ haloalkyl, or —OR⁵, or a pharmaceutically acceptable salt thereof.

18. The compound of any one of clauses 1 or clauses 14-17, wherein x is0, or a pharmaceutically acceptable salt thereof.

19. The compound of any one of clauses 1 or clauses 14-17, wherein x is1, 2, or 3, or a pharmaceutically acceptable salt thereof.

20. The compound of any one of clauses 1 or clause 14-19, wherein R³ isC₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to7-membered heterocycloalkyl, C₆-C₁₀ aryl, C₆-C₁₀ aryl-(C₆-C₁₀ aryl),C₁-C₆ alkyl-(NHR⁸), C₁-C₆ alkyl-(OR⁸), C₆-C₁₀ aryl-(OR⁸), C₁-C₆alkyl-(NR⁸R⁹), or mono- or bicyclic heteroaryl; wherein each hydrogenatom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3-to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, or mono- or bicyclicheteroaryl is optionally substituted by deuterium, halogen, —OH, oxo,—OR⁸, —NHR, —CN, —OC₁-C₆ alkyl, —NH₂, —NH(C₁-C₆ alkyl), —NH(C₆-C₁₀aryl), —NH(C₆-C₁₀ aryl)-N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl, —N(C₁-C₆alkyl)C(O)NH₂, —N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl, —NHC(O)N(C₁-C₆ alkyl)₂,—N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆alkyl)C(O)OC₁-C₆ alkyl, —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),—NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),—NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆ alkyl)₂, —CO₂H,—C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂,—SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆alkyl), —S(O)₂NH(C₁-C₆ alkyl), —S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆alkyl)₂, —P(C₁-C₆ alkyl)₂, —P(O)(C₁-C₆ alkyl)₂, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, —C₃-C₆ cycloalkyl, 3- to 7-memberedheterocycloalkyl, C₁-C₆ alkyl-(3- to 7-membered heterocycloalkyl), —CF₃,—CHF₂, or —CH₂F, or a pharmaceutically acceptable salt thereof.

21. The compound of any one of clauses 1 or clauses 14-20, wherein R³ isC₁-C₆ alkyl, C₂-C₆ alkenyl, C₆-C₁₀ aryl, C₁-C₆ alkyl-(OR⁸), C₆-C₁₀aryl-(OR⁸), or monocyclic heteroaryl; wherein each hydrogen atom inC₁-C₆ alkyl, C₂-C₆ alkenyl, C₆-C₁₀ aryl, or monocyclic heteroaryl isoptionally substituted by deuterium, halogen, —OH, oxo, —OR⁸, —NHR⁸,—CN, —OC₁-C₆ alkyl, —NH₂, —NH(C₁-C₆ alkyl), —NH(C₆-C₁₀ aryl), —NH(C₆-C₁₀aryl)-N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)C₁-C₆alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)NH₂,—N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl, —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆alkyl)C(O)OC₁-C₆ alkyl, —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),—NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),—NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆ alkyl)₂, —CO₂H,—C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂,—SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆alkyl), —S(O)₂NH(C₁-C₆ alkyl), —S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆alkyl)₂, —P(C₁-C₆ alkyl)₂, —P(O)(C₁-C₆ alkyl)₂, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, —C₃-C₆ cycloalkyl, 3- to 7-memberedheterocycloalkyl, C₁-C₆ alkyl-(3- to 7-membered heterocycloalkyl), —CF₃,—CHF₂, or —CH₂F, or a pharmaceutically acceptable salt thereof.

22. The compound of any one of clauses 1 or clauses 14-21, wherein R³ isC₁-C₆ alkyl, C₂-C₆ alkenyl, C₆-C₁₀ aryl, C₁-C₆ alkyl-(OR⁸), C₆-C₁₀aryl-(OR⁸), or monocyclic heteroaryl; wherein each hydrogen atom inC₁-C₆ alkyl, C₂-C₆ alkenyl, C₆-C₁₀ aryl, or monocyclic heteroaryl isoptionally substituted by deuterium, halogen, —OH, —OR⁸, —OC₁-C₆ alkyl,C₁-C₆ alkyl, or 3- to 7-membered heterocycloalkyl, or a pharmaceuticallyacceptable salt thereof.

23. The compound of clause 1, wherein the compound has the formula(III),

-   -   wherein R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        C₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C₁₀        aryl, or mono- or bicyclic heteroaryl; wherein each hydrogen        atom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆        cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, or        mono- or bicyclic heteroaryl is independently optionally        substituted by deuterium, halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl,        or —OR⁵;    -   R³ is independently H, deuterium, C₁-C₆ alkyl, C₂-C₆ alkenyl,        C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to 7-membered        heterocycloalkyl, C₆-C₁₀ aryl, C₆-C₁₀ aryl-(C₆-C₁₀ aryl), C₁-C₆        alkyl-(NHR⁸), C₁-C₆ alkyl-(OR⁸), C₆-C₁₀ aryl-(OR⁸), C₁-C₆        alkyl-(NR⁸R⁹), or mono- or bicyclic heteroaryl; wherein each        hydrogen atom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        C₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C₁₀        aryl, or mono- or bicyclic heteroaryl is optionally substituted        by deuterium, halogen, —OH, oxo, —OR⁸, —NHR⁸, —CN, —OC₁-C₆        alkyl, —NH₂, —NH(C₁-C₆ alkyl), —NH(C₆-C₁₀ aryl), —NH(C₆-C₁₀        aryl)-N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆        alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl,        —N(C₁-C₆ alkyl)C(O)NH₂, —N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl,        —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂,        —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)OC₁-C₆ alkyl,        —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl), —N(C₁-C₆        alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),        —NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆        alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),        —NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆        alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl),        —N(C₁-C₆ alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆        alkyl)₂, —CO₂H, —C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆        alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl,        —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl),        —S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆ alkyl)₂, —P(C₁-C₆ alkyl)₂,        —P(O)(C₁-C₆ alkyl)₂, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        —C₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl, C₁-C₆        alkyl-(3- to 7-membered heterocycloalkyl), —CF₃, —CHF₂, or        —CH₂F; and    -   each R⁵, R⁶, R⁷, R⁸ and R⁹ is independently H, deuterium, C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to        7-membered heterocycloalkyl, C₆-C₁₀ aryl, C₁-C₆ alkyl-3- to        7-membered heterocycloalkyl, heteroaryl, —S(O)₂C₁-C₆ alkyl,        —CF₃, —CHF₂, or —CH₂F, wherein each hydrogen in C₆-C₁₀ aryl is        optionally substituted by C₃-C₆ cycloalkyl, 3- to 7-membered        heterocycloalkyl, or —CH₂CN;    -   or a pharmaceutically acceptable salt thereof.

24. The compound of clause 1 or clause 23, wherein R¹ is H, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, or 3- to 7-memberedheterocycloalkyl, wherein each hydrogen atom in C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, or 3- to 7-memberedheterocycloalkyl is independently optionally substituted by deuterium,halogen, C1-C6 alkyl, C1-C6 haloalkyl, or —OR5, or a pharmaceuticallyacceptable salt thereof.

25. The compound of clause 1, clause 23, or clause 24, wherein R¹ is H,C₁-C₆ alkyl, C₃-C₆ cycloalkyl, or 3- to 7-membered heterocycloalkyl,wherein each hydrogen atom in C₁-C₆ alkyl, C₃-C₆ cycloalkyl, or 3- to7-membered heterocycloalkyl is independently optionally substituted bydeuterium, halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or —OR⁵, or apharmaceutically acceptable salt thereof.

26. The compound of any one of clauses 1 or clauses 23-25, wherein R¹ isH or C₁-C₆ alkyl, wherein each hydrogen atom in C₁-C₆ alkyl isindependently optionally substituted by deuterium, halogen, C₁-C₆ alkyl,C₁-C₆ haloalkyl, or —OR⁵, or a pharmaceutically acceptable salt thereof.

27. The compound of any one of clauses 1 or clauses 23-26, wherein R³C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to7-membered heterocycloalkyl, C₆-C₁₀ aryl, C₆-C₁₀ aryl-(C₆-C₁₀ aryl),C₁-C₆ alkyl-(NHR⁸), C₁-C₆ alkyl-(OR⁸), C₆-C₁₀ aryl-(OR⁸), C₁-C₆alkyl-(NR⁸R⁹), or mono- or bicyclic heteroaryl; wherein each hydrogenatom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3-to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, or mono- or bicyclicheteroaryl is optionally substituted by deuterium, halogen, —OH, oxo,—OR⁸, —NHR⁸, —CN, —OC₁-C₆ alkyl, —NH₂, —NH(C₁-C₆ alkyl), —NH(C₆-C₁₀aryl), —NH(C₆-C₁₀ aryl)-N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl, —N(C₁-C₆alkyl)C(O)NH₂, —N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl, —NHC(O)N(C₁-C₆ alkyl)₂,—N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆alkyl)C(O)OC₁-C₆ alkyl, —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),—NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),—NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆ alkyl)₂, —CO₂H,—C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂,—SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆alkyl), —S(O)₂NH(C₁-C₆ alkyl), —S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆alkyl)₂, —P(C₁-C₆ alkyl)₂, —P(O)(C₁-C₆ alkyl)₂, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, —C₃-C₆ cycloalkyl, 3- to 7-memberedheterocycloalkyl, C₁-C₆ alkyl-3- to 7-membered heterocycloalkyl, —CF₃,—CHF₂, or —CH₂F, or a pharmaceutically acceptable salt thereof.

28. The compound of any one of clauses 1 or clauses 23-27, wherein R³ isC₁-C₆ alkyl, C₂-C₆ alkenyl, C₆-C₁₀ aryl, C₁-C₆ alkyl-(OR⁸), C₆-C₁₀aryl-(OR⁸), or monocyclic heteroaryl; wherein each hydrogen atom inC₁-C₆ alkyl, C₂-C₆ alkenyl, C₆-C₁₀ aryl, or monocyclic heteroaryl isoptionally substituted by deuterium, halogen, —OH, oxo, —OR⁸, —NHR⁸,—CN, —OC₁-C₆ alkyl, —NH₂, —NH(C₁-C₆ alkyl), —NH(C₆-C₁₀ aryl), —NH(C₆-C₁₀aryl)-N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)C₁-C₆alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)NH₂,—N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl, —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆alkyl)C(O)OC₁-C₆ alkyl, —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),—NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),—NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆ alkyl)₂, —CO₂H,—C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂,—SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆alkyl), —S(O)₂NH(C₁-C₆ alkyl), —S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆alkyl)₂, —P(C₁-C₆ alkyl)₂, —P(O)(C₁-C₆ alkyl)₂, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, —C₃-C₆ cycloalkyl, 3- to 7-memberedheterocycloalkyl, C₁-C₆ alkyl-3- to 7-membered heterocycloalkyl, —CF₃,—CHF₂, or —CH₂F, or a pharmaceutically acceptable salt thereof.

29. The compound of any one of clauses 1 or clauses 23-28, wherein R³ isC₁-C₆ alkyl, C₂-C₆ alkenyl, C₆-C₁₀ aryl, C₁-C₆ alkyl-(OR⁸), C₆-C₁₀aryl-(OR⁸), or monocyclic heteroaryl; wherein each hydrogen atom inC₁-C₆ alkyl, C₂-C₆ alkenyl, C₆-C₁₀ aryl, or monocyclic heteroaryl isoptionally substituted by deuterium, halogen, —OH, —OR⁸, —OC₁-C₆ alkyl,C₁-C₆ alkyl, or 3- to 7-membered heterocycloalkyl, or a pharmaceuticallyacceptable salt thereof.

30. The compound of clause 1, wherein the compound is of the formula

or a pharmaceutically acceptable salt thereof.

31. A process for preparing a compound of the formula (I),

-   -   wherein A and B are independently H, deuterium, halogen, C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₆-C₁₀        aryl, —OH, —CN, —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂,        —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)C₁-C₆ alkyl, —OR⁴, CO₂H,        —C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆        alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl,        —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl); or A and B taken        together with the ring to which they are attached form a C₅-C₈        cycloalkyl, C₆-C₁₀ aryl, or a 5- to 8-membered heterocycloalkyl;        wherein each hydrogen atom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, C₃-C₆ cycloalkyl, C₅-C₈ cycloalkyl, C₆-C₁₀ aryl, or 5-        to 8-membered heterocycloalkyl is independently optionally        substituted by deuterium, halogen, —OH, —CN, —OR⁴, —OC₁-C₆        alkyl, —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆        alkyl, —N(C₁-C₆ alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂,        —NHC(O)NHC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)NH₂, —N(C₁-C₆        alkyl)C(O)NHC₁-C₆ alkyl, —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆        alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆        alkyl)C(O)OC₁-C₆ alkyl, —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆        alkyl), —N(C₁-C₆ alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆        alkyl)S(O)₂(C₁-C₆ alkyl), —NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆        alkyl)S(O)NH₂, —N(C₁-C₆ alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl),        —NHS(O)₂NH(C₁-C₆ alkyl), —NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆        alkyl)₂, —N(C₁-C₆ alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆        alkyl)S(O)₂NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)N(C₁-C₆ alkyl)₂,        —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆ alkyl)₂, —CO₂H, —C(O)OC₁-C₆ alkyl,        —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆        alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆        alkyl), —S(O)₂NH(C₁-C₆ alkyl), —S(O)N(C₁-C₆ alkyl)₂,        —S(O)₂N(C₁-C₆ alkyl)₂, —P(C₁-C₆ alkyl)₂, —P(O)(C₁-C₆ alkyl)₂,        —C₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl, —CF₃,        —CHF₂, or —CH₂F;    -   R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆        cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, or        mono- or bicyclic heteroaryl; wherein each hydrogen atom in        C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3-        to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, or mono- or        bicyclic heteroaryl is independently optionally substituted by        deuterium, halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or —OR⁵;    -   each R² is independently deuterium, halogen, —OH, —CN, C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₆-C₁₀        aryl, —OR⁶, —C(O)OR⁶, —C(O)NR⁶R⁷, —NH₂, —NH(C₁-C₆ alkyl),        —N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —CO₂H, —C(O)OC₁-C₆ alkyl,        —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆        alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆        alkyl), —S(O)₂NH(C₁-C₆ alkyl); wherein each hydrogen atom in        C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl and        C₆-C₁₀ aryl is independently optionally substituted by        deuterium, halogen, —OH, —CN, —OR⁶, —OC₁-C₆ alkyl, —NH₂,        —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆        alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl,        —N(C₁-C₆ alkyl)C(O)NH₂, —N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl,        —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂,        —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)OC₁-C₆ alkyl,        —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl), —N(C₁-C₆        alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),        —NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆        alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),        —NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆        alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl),        —N(C₁-C₆ alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆        alkyl)₂, —CO₂H, —C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆        alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl,        —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl),        —S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆ alkyl)₂, —P(C₁-C₆ alkyl)₂,        —P(O)(C₁-C₆ alkyl)₂, —C₃-C₆ cycloalkyl, or 3- to 7-membered        heterocycloalkyl;    -   R³ is independently H, deuterium, C₁-C₆ alkyl, C₂-C₆ alkenyl,        C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to 7-membered        heterocycloalkyl, C₆-C₁₀ aryl, C₆-C₁₀ aryl-(C₆-C₁₀ aryl), C₁-C₆        alkyl-(NHR⁸), C₁-C₆ alkyl-(OR⁸), C₆-C₁₀ aryl-(OR⁸), C₁-C₆        alkyl-(NR⁸R⁹), or mono- or bicyclic heteroaryl; wherein each        hydrogen atom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        C₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C₁₀        aryl, or mono- or bicyclic heteroaryl is optionally substituted        by deuterium, halogen, —OH, oxo, —OR⁸, —NHR⁸, —CN, —OC₁-C₆        alkyl, —NH₂, —NH(C₁-C₆ alkyl), —NH(C₆-C₁₀ aryl), —NH(C₆-C₁₀        aryl)-N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆        alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl,        —N(C₁-C₆ alkyl)C(O)NH₂, —N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl,        —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂,        —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)OC₁-C₆ alkyl,        —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl), —N(C₁-C₆        alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),        —NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆        alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),        —NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆        alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl),        —N(C₁-C₆ alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆        alkyl)₂, —CO₂H, —C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆        alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl,        —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl),        —S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆ alkyl)₂, —P(C₁-C₆ alkyl)₂,        —P(O)(C₁-C₆ alkyl)₂, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        —C₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl, C₁-C₆        alkyl-(3- to 7-membered heterocycloalkyl), —CF₃, —CHF₂, or        —CH₂F;    -   each R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ is independently H, deuterium,        C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3-        to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, C₁-C6 alkyl-(3- to        7-membered heterocycloalkyl), heteroaryl, —S(O)₂C₁-C₆ alkyl,        —CF₃, —CHF₂, or —CH₂F, wherein each hydrogen in C₆-C₁₀ aryl is        optionally substituted by C₃-C₆ cycloalkyl, 3- to 7-membered        heterocycloalkyl, or —CH₂CN; and    -   x is 0, 1, 2, or 3,    -   the process comprising contacting a compound of the formula (V),

wherein A, B, R¹, R², and x are as defined in formula (I), with acompound of the formula (VI),

wherein R³ is as defined in formula (I), Y is absent or a halogen, and Mis a metal, or with a compound of the formula (VIa),

wherein R³ is as defined in formula (I) and a catalyst.

32. The process of clause 31, wherein the compound of formula (VI),

is prepared by contacting a compound of the formula (VIa),

with a compound of the formula (VII),R^(B)YX  (VII)

wherein R^(B) is C₁-C₆ alkyl, M is a metal, and Y is a halogen.

33. The process of clause 31 or clause 32, wherein M is magnesium.

34. The process of any one of clauses 31-33, wherein Y is bromo orchloro.

35. The process of clause 31, wherein Y is absent and M comprises zinc.

36. The process of clause 35, wherein the zinc is diethyl zinc.

37. The process of anyone of clauses 31-36, wherein the process furthercomprises a base.

38. The process of clause 37, wherein the base is N-methylimidazole.

39. The process of clause 31, wherein the catalyst is a rutheniumcatalyst.

40. The process of clause 31 or clause 39, wherein the catalyst is(Cp*RuCl)₄.

41. The process of any one of clauses 31-40, wherein the step ofcontacting is performed in the presence of a polar solvent.

42. The process of clause 41, wherein the polar solvent is THF, DMF,dichloromethane, Et₂O, diglyme, or a mixture thereof.

43. The process of any one of clauses 31-42, wherein the process iscarried out at an elevated temperature.

44. The process of clause 43, wherein the elevated temperature is atleast 35° C.

45. The process of clause 44, wherein the elevated temperature isselected from a range of about 35° C. to about 65° C.

46. The process of any one of clause 31-45, wherein the compound of theformula (V),

is prepared by

-   -   (a) contacting a compound of the formula (VIII),

-   -   with an acid in an aqueous solvent to form an intermediate, and    -   (b) reacting the intermediate formed in (a) with an azide.

47. The process of clause 46, wherein the acid is a mineral acid.

48. The process of clause 47, wherein the mineral acid is sulfuric acid.

49. The process of any one of clauses 46-48, wherein the azide is sodiumazide.

50. The process of any one of clauses 46-49, wherein step (a) furthercomprises sodium nitrite.

51. The process of any one of clauses 46-50, further comprisingpurifying the compound of formula (V) by chromatography.

52. The process of any one of clauses 31-51, wherein A and B are takentogether with the ring to which they are attached form a C₆-C₁₀ aryl.

53. The process of any one of clauses 31-52, wherein the compound offormula (I) has the structure of a compound of the formula (III),

wherein R¹ and R³ are defined as in clause 31, and the compound offormula (V) has the structure of formula (Va),

54. The process of clause 53, wherein the process comprises the step ofcontacting the compound of the formula (Va),

with a compound of the formula VI,

wherein R³ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆cycloalkyl, 3- to 7-membered heterocycloalkyl, C₁-C₆ alkyl-(OR⁸), orC₁-C₆ alkyl-(NR⁸R⁹), Y is absent and M is a metal, or with a compound ofthe formula VIa,

wherein R³ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆cycloalkyl, 3- to 7-membered heterocycloalkyl, C₁-C₆ alkyl-(OR⁸), orC₁-C₆ alkyl-(NR⁸R⁹) and a catalyst.

55. The process of clause 54, wherein M comprises zinc.

56. The process of clause 55, wherein the zinc is diethyl zinc.

57. The process of any one of clauses 54-56, wherein the process furthercomprises a base.

58. The process of clause 57, wherein the base is N-methylimidazole.

59. The process of clause 54, wherein the catalyst is a rutheniumcatalyst.

60. The process of clause 54 or clause 59, wherein the catalyst is(Cp*RuCl)₄.

61. The process of clause 54, wherein the compound of the formula (Va),

is contacted by a compound of the formula (VIa),

and a catalyst.

62. The process of clause 61, wherein the catalyst is a rutheniumcatalyst.

63. The process of clause 61, wherein the catalyst is (Cp*RuCl)₄.

64. The process of clause 54, wherein the compound of the formula (Va),

is contacted by a compound a compound of the formula (VI),

wherein Y is absent and M is a metal.

65. The process of clause 64, wherein M comprises zinc.

66. The process of clause 65, wherein the zinc is diethyl zinc.

67. The process of any one of clauses 64-66, wherein the process furthercomprises a base.

68. The process of clause 67, wherein the base is N-methylimidazole.

69. The process of clause 53, wherein the process comprises the step ofcontacting the compound of the formula (Va),

with a compound of the formula (VI),

wherein R³ C₆-C₁₀ aryl, C₆-C₁₀ aryl-(C₆-C₁₀ aryl), C₆-C₁₀) aryl-(OR⁸),or mono- or bicyclic heteroaryl, Y is a halogen, and M is a metal.

70. The process of clause 69, wherein M is magnesium.

71. The process of clause 69 or clause 70, wherein Y is bromo or chloro.

72. The process of any one of clauses 69-71, wherein the process isperformed in a polar solvent selected from the group consisting of THF,DMF, dichloromethane, Et₂O, diglyme, and a mixture thereof.

73. The process of any one of clauses 69-72, wherein the process iscarried out at an elevated temperature.

74. The process of clause 73, wherein the elevated temperature is atleast 35° C.

75. The process of clause 73 or clause 74, wherein the elevatedtemperature is selected from a range of about 35° C. to about 65° C.

76. A method of treating a disease in a patient, the method comprisingadministering to the patient in need thereof an effective amount of acompound according to any one of clauses 1-30.

77. The method of clause 76, wherein the disease is cancer.

78. The method of clause 76 or clause 77, wherein the compound inhibitscancer cell migration.

79. The method of clause 77 or clause 78, wherein the cancer is selectedfrom the group consisting of breast cancer, pancreatic cancer, ovariancancer, gastric cancer, and neuronal cancer.

80. The method of clause 79, wherein the cancer is pancreatic cancer.

81. The method of clause 79, wherein the cancer is ovarian cancer.

82. The method of clause 79, wherein the cancer is gastric cancer.

83. The method of clause 79, wherein the cancer is neuronal cancer.

84. The method of clause 79, wherein the cancer is breast cancer.

85. The method of any one of clauses 76-79 or clause 84, wherein thecompound inhibits mammosphere formation.

86. The method of any one of clauses 76-85, wherein the compound inducescell cycle arrest of a diseased cell.

87. The method of any one of clauses 76-86, wherein the compound inducesapoptosis of a diseased cell.

88. The method of any one of clauses 76-87, wherein the compound reducesthe expression of a Bcl-2 protein.

89. The method of any one of clauses 76-88, wherein the disease ismediated by a GTPase.

90. The method of clause 89, wherein the GTPase is Rac 1 or Cdc42.

91. The method of clause 89, wherein the GTPase is Rac1.

92. The method of clause 89, wherein the GTPase is Cdc42.

93. The method of any one of clauses 76-92, wherein the compoundinhibits PAK1/2 activity.

94. The method of any one of clauses 76-93, wherein the compoundinhibits STAT3 activity.

95. The method of any one of clauses 76-94, wherein the compound is ofthe formula

or a pharmaceutically acceptable salt thereof.

96. The method of any one of clauses 76-95, wherein the effective amountof the compound is in a range of about 0.01 mg/kg to about 100 mg/kg ofbody weight of the patient.

97. The method of any one of clauses 76-95, wherein the effective amountof the compound is in a range of about 0.1 mg/kg to about 50 mg/kg ofbody weight of the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Design and synthesis of MBQ-167. Synthesis of MBQ-167. Reactionconditions: (i) conc. H₂SO₄, NaNO₂, water 0-5° C., 1 h; (ii) NaN₃, 0°C., 1 h, 76%; (iii) THF, 3, 50° C., 1 h; (iv) NH₄Cl (aq), 86%.

FIG. 2(A, B, C). Breast cancer cell phenotype following MBQ-167treatment. 2A, Upper panel, bright field images of MDA-MB-231 humanmetastatic breast cancer cells in response to MBQ-167. Lower panel,MCF-7 cells in response to MBQ-167. 2B, C, Focal adhesions and actincytoskeleton following MBQ-167 treatment showing representativefluorescence micrographs of MDA-MB-231 cells. MDA-MB-231 cells weretreated with 0, 250, or 500 nM MBQ-167 for 24 h and fixed and stained in2B, with phospho-tyrosine antibodies for focal adhesions (green) andRhodamine phalloidin for F-actin (red) and in 2C, with anti vinculin(red). Arrows, invadopodia; arrowheads, focal adhesions.

FIG. 3. (A, B, C). Inhibitory effect of MBQ-167 on Rac and Cdc42activation. MDA-MB-231 human breast cancer cells were treated for 24 hwith 250 nM MBQ-167. A-C, The attached (A) and detached (D) cellpopulations were recovered and equal amounts of proteins subjected topulldown assays using the p21-binding domain of PAK to isolate the GTPbound Rac and Cdc42. Cell lysates were western blotted with antibodiesto Rac or Cdc42. Results from positive bands in western blots werequantified using image J. 3A-1, Left, Representative western blot forRac1/2/; 3A-2 right, quantification of Rac activation at 24 h following0 or 250 nM MBQ-167. 3B-1, Left, Representative western blot for Cdc42;3B-2 right, quantification of Cdc42 activation following 24 h treatmentwith 0 or 250 nM MBQ-167. The integrated density for active Rac or Cdc42(GTP) was divided by the total Rac or Cdc42 from the same cell lysates.Rac or Cdc42 activity for each MBQ-167 treatment was divided by thevehicle controls for each experiment to obtain Relative Rac or Cdc42activity. N=3, *=P<0.05, ***=P<0.001. Error bars represent ±S.E.M. C,D,MDA-MB-231 cells with vehicle control (0.1% DMSO) or varyingconcentrations of MBQ-167 (0-1000 nM) were treated for 24 hrs. Totalcell lysates using combined attached and detached treated populationswere subjected to the G-LISA Rac1/2/3 or Cdc42 activation assay. IC₅₀curves for percentage Rac 3C or Cdc42 3D activation are relative tovehicle from three biological replicates each with two technicalreplicates. Error bars represent ±S.D. Four-parameter dose-responsecurves generated using GraphPad Prism® are shown.

FIG. 4 (A-G). The effect of MBQ-167 on signaling downstream of Rac andCdc42.4A, The effect of MBQ-167 on PAK1 and PAK2 phosphorylation asmeasured by western blotting for pPAK1 (T423)/pPAK2 (T402), pPAK1(S199)/pPAK2 (S192), and pPAK1 (S144) levels in MDA-MB-231 cells after24 hours of treatment in 0 or 250 nM MBQ-167. Data for separate attached(Att) and detached (Det) populations are shown. 4A-1 Left,representative western blots (N=3) 4A-2. Right, relative PAK activityfollowing MBQ-167 treatment. Positive bands from all western blots werequantified using image J. The integrated density of p-PAK was divided bythat of total PAK for the same cell lysate and used as a measure of PAKactivity for each phospho PAK residue. Relative PAK activity wasdetermined relative to vehicle controls for each experiment. N=3,*=P<0.05, **=P<0.01, ***=P<0.001. Error bars represent ±S.E.M. 4B,Effect of MBQ-167 on PAK downstream effectors LIMK and cofilinphosphorylation. MDA-MB-231 cells were incubated for 4, 12, or 24 h invehicle or 250 nM MBQ-167, the attached (A) and detached (D) populationswere separated, lysed, and equal protein used for western blotting.Representative western blot of total LIMK1 or p-LIMK1/2 (Y507/T508)following 24 h in 0 or 250 nM MBQ-167 (N=2) is shown. 4C, Representativewestern blot of total or p-cofilin (S3) of equal amounts of totalprotein lysates following 4, 12, or 24 h in 250 nM MBQ-167 (N=3).Separated attached (A) and detached (D) populations are shown for 12 and24 h of MBQ-167 treatment. 4D, Effect of MBQ-167 on STAT3phosphorylation and expression. Representative western blot is shown forpSTAT3 (Y705) and total STAT3 expression in GFP-HER2-BM cells after 24 htreatment with vehicle or 100, 200, or 500 nM MBQ-167. 4D-1Representative western blot (left) 4D-2 and quantification (right). N=3,*=P<0.05, Error bars represent ±S.E.M. E, F, Effect of MBQ-167 on cellmigration. 4E-1, The effect of MBQ-167 on MDA-MB-231 cellular migrationas measured by a transwell assay. Images are representative of threeindependent experiments. 4E-2 The Graph below shows quantification of 20microscopic fields per treatment per experiment of PI stained cells thatmigrated to the underside of the membrane through 8 micron diameterpores N=3, *=P<0.05, Error bars represent ±S.E.M. 4F, The effect ofMBQ-167 on cell migration in a scratch assay. MDA-MB-231 cells plated atequal density were subjected to a scratch in the center and treated withMBQ-167 at 0, 250, or 500 nM. Micrographs were digitally acquired at 0and 24 h and the distance of the scratch quantified for each treatmentand presented relative to the distance at time 0. Results are an averageof two technical replicates and two biological replicates for eachtreatment ±S.D, *=P<0.05. 4G, The effect of MBQ-167 on mammosphereforming efficiency in MDA-MB-231 cells. MDA-MB-231 cells treated with 0or 250 nM MBQ-167 were subjected to mammosphere assays for 4 days. Cellswere treated with MBQ-167 only once prior to placing on the mammospheremedium. Mammosphere forming efficiency was calculated as the percentageof the number of mammospheres divided by the number of cells seeded perwell. N=3, ***=P<0.001, Error bars represent ±S.D.

FIG. 5A-D. Effect of MBQ-167 on cell survival. 5A, The effect of MBQ-167on MDA-MB-231, GFP-HER2-BM, and MCF10A cell viability. Equal numbers ofeach respective cell line were treated with vehicle control (0.1% DMSO)or varying concentrations of MBQ-167 (0-1000 nM) for 120 h. GI₅₀ curvesfor percentage cell viability are relative to vehicle from threebiological replicates each with two technical replicates. Four-parameterdose-response curves generated using GraphPad Prism® are shown. N=3,Error bars represent ±S.E.M. 5B, The effect of MBQ-167 on cell cycleprogression. Equal numbers of MDA-MB-231 cells in either vehicle controlor treatment groups were treated for 24 h with 0 or 250 nM MBQ-167.Graphs represent the percentage of control versus 250 nM MBQ-167 treatedcells stained with PI in G₀/G₁, S, or G₂/M phases of the cell cycle.5B-1 Left graph, representative flow cytometry analysis 5B-2; rightgraph, quantification of cell cycle stage. N=3, Error bars ±S.E.M. 5C,The effect of MBQ-167 on caspase3,-7 activity. 5C-1 Left graph,caspase3/7 activity of MDA-MB-231 and GFP-HER2-BM cells (includingattached and detached populations) following vehicle (0.1% DMSO) orvarying concentrations of MBQ-167 (0-1000 nM) for 24 h. N=3, Error barsrepresent ±S.E.M. 5C-2 Right graph, the effect of MBQ-167 oncaspase-3,-7 activity of MDA-MB-231 cells. Cells were treated with 250nM MBQ-167 for 24 h and equal numbers of separated attached and detachedcells were lysed and used for caspase 3/7 assays. Caspase-3,-7 activityrelative to equal number of attached cells from control cells is shown.N=3, Error bars represent ±S.E.M. 5D, The effect of MBQ-167 onmitochondrial regulation of apoptosis. The effect of MBQ-167 on theexpression of the pro-survival proteins Bcl-2, Bcl-xL, and Mcl-1 inMDA-MB-231 cells after 24 h of treatment. 5D-1 Left panel,representative western blot 5D-2; Right panel, quantification of theintegrated density of positive bands using image J. N=3, *=P<0.05,***=P<0.001, Error bars represent ±S.E.M.

FIG. 6 (A-E). In-vivo efficacy of MBQ-167 in HER2-type breast cancer.Mammary fatpad tumors were established in nude mice by inoculating 5×10⁵GFP-HER2-BM cells. Following one week, mice were treated with vehiclecontrol or 1.0, or 10.0 mg/kg body weight (BW) MBQ-167 3× a week by i.p.injection. 6A-1, Left panel, representative excised tumors following 0,1, 10 mg/kg BW MBQ-167. 6A-2 Right panel, average relative tumor growthfrom fluorescence in situ images up to 65 days following 0, 1.0, or 10mg/kg BW MBQ-167 (3× a wk) (N=6). B, Representative fluorescencemicrographs of lungs, spleens, and kidneys from vehicle or MBQ-167treated mice following necropsy. 6C, Mouse weights from 1-65 days. 6D,E, Liver enzyme activities following MBQ-167 treatment. Followingnecropsy, livers were harvested, lysed and subjected to 6D, ALP activityor 6E, ALT activity assays. N=4, error bars represent ±S.E.M.

FIG. 7. The effect of MBQ-167 on the Rac activity of metastatic cancercells expressing a constitutively active Rac (Rac1G12V). MDA-MB-435human metastatic cancer cells expressing wild type Rac1 (WT) orRac1(G12V) constitutively activated form were were treated for 24 h with250 nM MBQ-167. The attached (A) and detached (D) cell populations wererecovered and equal amounts of proteins subjected to pulldown assaysusing the p21-binding domain of PAK to isolate the GTP bound Rac. Celllysates were western blotted with antibodies to Rac. Results frompositive bands in western blots were quantified using image J. Resultsshow that even though MBQ-167 significantly inhibited the Rac activityof the detached cells expressing wild type Rac, this inhibition was notobserved in cells expressing a constitutively active GTP bound form ofRac1.

FIG. 8(A, B). The effect of MBQ-167 on survival and PAK activity inNCI-N87 gastric cancer cells. 8A. Equal numbers of NCI-N87 cells ineither vehicle control or treatment groups were treated for 24 h with 0or 250 nM MBQ-167. Cells were subjected to MTT assays for viability.Results show that 250 nM MBQ-167 for 24 h reduces NCI-N87 cell viabilityby 50%. 8B. Equal numbers of cells treated with 0, 250, or 500 nMMBQ-167 were lysed and western blotted for phospho (P)-PAK1^(T432)(active form) or total PAK1, or phospho (P)-PAK4^(S474) (active form) ortotal PAK4. Representative western blot shown demonstrates that MBQ-167inhibts both PAK1 and PAK4 activities in the NCI-N87 gastric cancercells.

FIG. 9. Human cancer cell phenotype following MBQ-167 treatment. Triplenegative breast cancer (MDA-MB-468), aggressive pancreatic (Mia PaCa-2),ovarian (SKVO3), gastric carcinoma (AGS and NCI-N87) cell lines and aneuroblastoma (SH—SY5Y) cell line were treated with 0, 250 or 500 nMMBQ-167 for 24 h. Representative bright field images are shown for eachcell line.

FIG. 10 (A-C). Effect of MBQ-167 on Rho GTPase activity in GFP-HER2-BMcells. GFP-HER2-BM cells were treated for 24 h with 250 nM MBQ-167. Theattached and detached cell populations were recovered and equal amountsof proteins subjected to G-LISA assays for 10A Rac or Cdc42 10B, 10CGFP-HER2-BM human breast cancer cells were treated with 0 or MBQ-167 for24 h were lysed (both attached and detached cells) and incubated withthe Rho-binding domain from Rhotekin to isolate active Rho. Cell lysateswere western blotted with antibodies to Rac, Cdc42, or Rho. Results frompositive bands in western blots were quantified using image J. Theintegrated density for active Rho was divided by the total Rho from thesame cell lysates. Rac, Cdc42, or Rho activity for each MBQ-167treatment was divided by the vehicle controls for each experiment toobtain Relative activity.

FIG. 11. MBQ-167 does not affect PAK activity in MCF-7 cells. MCF-7cells were treated with 0 or 250 nM MBQ-167 for 24 h and the attached(A) and detached (D) cells were recovered separately. Equal amounts ofprotein were run on SDS-PAGE and western blotted with a phospho-PAK(T423) or total PAK antibody. Representative western (N=2) is shown.

FIG. 12 (A, B). Effect of MBQ-167 on STAT3 activation and cell migrationin GFP-HER2-BM metastatic cancer cells. 12A, The effect of MBQ-167 onPAK1 and PAK2 phosphorylation as measured by western blotting for STAT3and p-STAT3 (Y705) levels in MDA-MB-231 and GFP-HER2-BM cells after 24hours of treatment in 0 or 250 nM MBQ-167. Data for separate attached(A) and detached (D) populations are shown. Representative western blots(N=2). 12B, The effect of MBQ-167 on GFP-HER2-BM cell migration asmeasured by a Transwell assay. The Graph shows quantification of 20microscopic fields per treatment per experiment of PI stained cells thatmigrated to the underside of the membrane through 8 micron diameterpores in 6 h, N=3, *=P<0.05, Error bars represent ±S.E.M.

FIG. 13 (A, B). MAPK and Akt signaling in MDA-MB-231 cells. MDA-MB-231cells were treated with 250 nM MBQ-167 for 24 h and equal amounts ofprotein from attached and detached cell lysates were western blotted for13A, phospho (active) p-38 and p-42/44 (ERK) MAPKs or 13B, total andphospho (s-473) Akt. Representative western blots of N=3 are shown.

FIG. 14. Annexin V staining in response to MBQ-167. GFP-MDA-MB-231 cellsgrowing on coverslips were treated with vehicle or MBQ-167 at 250 or 500nM for 6 h. Cells were fixed in formaldehyde and stained with AnnexinV-Cy3-18. Representative fluorescence micrographs are shown. Arrowindicates red fluorescent Annexin V staining at the membranes ofapoptotic cells.

FIG. 15 (A-D). Effect of MBQ-167 on bud polarity in Saccharomycescerevisiae. A haploid derivative of the yeast strain BY4741 expressingthe tTA transactivator, under the control of the CMV promoter, was usedto integrate into the Cdc42 promoter to conditionally knockout theessential Cdc42 gene expression. 15A Top left, representative micrographof the budding phenotype in the absence of Doxycycline. 15B Top right,representative micrograph of same cell strain following 24 h in 25 μMMBQ-167. 15C Bottom left, representative micrograph of cells following10 μg/ml doxycycline to knockdown Cdc42. 15D Bottom right,representative micrograph of cells with both Cdc42 knockdown and 25 μMMBQ-167.

FIG. 16. In vivo efficacy of MBQ-167 in triple negative breast cancer.Mammary fat pad tumors were established in severe combinedimmunodeficiency (SCID) mice using green fluorescent protein (GFP)tagged-MDA-MB-231 breast cancer cells. One week later, mice were treatedwith 0, 1.0, 5.0, or 10.0 mg/kg body weight (BW) MBQ-167 byintraperitoneal injections 3× a week for 50 days. Mammary tumor growthwas quantified from GFP image analysis. Results show average tumorgrowth in 50 days as a function of the size of each tumor on day 01.Vehicle control (0 mg/kg BW MBQ-167) is represented as 100% tumorgrowth. Administration of 1.0 mg/kg BW of MBQ-167 resulted in a 90%inhibition of tumor growth, 5.0 mg/kg BW treatment resulted in a 95%inhibition of tumor growth and the 10 mg/kg BW MBQ-167 treatmentresulted in 100% inhibition of tumor growth in 50 days.

DETAILED DESCRIPTION

Two compounds that inhibit Rho GTPases, Rac and Cdc42, have also beenused as drug targets. NSC23766 was the first Rac inhibitor shown toblock the interaction of Rac with the GEFs Trio and Tiam1; however, itshigh effective concentrations (IC₅₀>75 μM) limits its therapeutic use. Apanel of putative Rac and Cdc42 inhibitors led to the identification ofEHop-016. EHop-016 blocks the interaction of the GEF Vav2 with Rac, andinhibits Rac activity at an IC50 of ˜1.1 μM, which makes it ˜100× morepotent than NSC23766. EHop-016 also inhibits Cdc42 activity atconcentrations of ≥10 μM, without affecting Rho activity.

At 25 mg/kg body weight (BW), EHop-016 reduces mammary tumor growth,metastasis, and angiogenesis without apparent toxicity in nude mice. Thepharmacokinetics analysis of EHop-016, after oral and intraperitoneal(i.p.) administration, demonstrated a bioavailability of ˜30% with anaverage half-life of ˜4.5 h, indicating its potential as a cancertherapeutic in breast cancer, and subsequently in other types of cancer.

Although other small molecule inhibitors, such as the NSC23766derivative Aza-1 (inhibits both Rac and Cdc42) and CID2950007/ML141(selective for Cdc42) are currently available, they are effective in themicromolar range. A goal was to develop a Rac/Cdc42 inhibitor withimproved activities led to the identification of MBQ-167. Compared toEHop-016, MBQ-167 is a 10× more potent inhibitor of Rac and a 100× morepotent inhibitor of Cdc42, which resulted in an enhanced inhibition ofcancer malignancy.

Substituents A and B can alternatively form a ring system:

Wherein:

R5 is independently in each occurrence hydrogen, halide, hydroxyl,cyano, C1-6 alkyl, C3-6 cycloalkyl, C1-6 haloalkyl, nitro, amino, etc.

A compound according to formula I, includes one or more compoundsselected from the compounds in TABLE I.

There is a method for inhibiting Rac and Cdc42 activity in a cell bytreatment with compound I, wherein R1, R2, R3, R4, R5, Ra, Rb, and A andB are defined as above.

There is a method for inhibiting Rac and Cdc42 activity in a patientwith cancer of hyperproliferative disorder by treatment with compound I,wherein R1, R2, R3, R4, R5, Ra, Rb, and A and B are defined as above.

Compounds and Preparation

The 1,5-disubstituted 1,2,3-triazoles can be prepared in a two-stepprocess according to Scheme A, comprising step 1) formation of the azidefrom the corresponding amine; and step 2) formation of the 1,5-triazoleby reacting the azide with an alkyne in one of the conditions describedbelow.

Step 1 can be carried out by contacting a slurry of the appropriatearomatic amine in water with concentrated sulfuric acid, followed by thedropwise addition of an aqueous solution of sodium nitrite. Followingcompletion of the formation of the intermediate diazo-derivative, anaqueous solution of sodium azide is added, providing the azide productof step 1.

Step 2 can be carried out under various conditions that selectivelyprovide the 1,5-substituted 1,2,3-triazole products (in contrast withthe more commonly prepared 1,4-substituted 1,2,3-triazole product). Themost commonly employed methods in the literature include:

a) Contacting the primary alkyne with a Grignard reagent such asethylmagnesium bromide, which provides the alkynylmagnesium bromide.Subsequently reacting the alkynylmagnesium bromide product with theazide from step 1 selectively gives the 1,5-disubstituted 1,2,3-triazole

b) Contacting the primary alkyne with the azide in the presence ofpentamethylcyclopentadienyl ruthenium chloride regioselectively leads tothe 1,5-disubstituted 1,2,3-triazole

c) Contacting the primary alkyne with the azide in the presence ofdiethyl zinc and N-methylimidazole.

Many of the aromatic amine and alkyne starting materials arecommercially available or could be prepared via published procedures.Alternatively, modifications can be introduced after the cyclizationreaction has been performed. In some cases, protecting group strategiesneed to be applied.

Compounds are disclosed that inhibit RhoGTPases and therefore are usefulfor inhibiting hyperprofilerative and neoplastic diseases. Specifically,the compounds inhibit the GTPases Rac and Cdc42 that are overactive oroverexpressed in signaling pathways in cancer and metastasis. Thesecompounds are useful for the treatment of cancer and hyperproliferativediseases.

A series of 1,5-disubstituted 1,2,3-triazoles as novel inhibitors of Racand Cdc42. One specific example (MBQ-167) (previously EHop-167) wasstudied in extensive detail.

MBQ-167 Inhibits Rac and Cdc42 Activation in Metastatic Cancer Cells

MDA-MB-231 and GFP-HER2-BM cells were treated for 24 h with vehicle orMBQ-167, and the detached and attached cells (˜50% for each population),were recovered and lysed immediately. Equal amounts of protein weresubjected to activation assays for Rac or Cdc42. Following treatmentwith 250 nM MBQ-167 for 24 h, the attached population of MDA-MB-231cells demonstrated a ˜25% decrease in Rac activation while the detachedcells were more responsive with a ˜75% decrease (FIG. 3A). At earliertimes (6 h), treatment with 250 or 500 nm MBQ-167, induced a 10-20%inhibition in Rac activity in the attached cell population, while thedetached population demonstrated a ˜40-50% inhibition. Similarly, Cdc42activity was inhibited by 60% in the attached cells and 78% in thedetached MDA-MB-231 cells following 250 nM MBQ-167 for 24 h (FIG. 3C).These results indicate that both Rac and Cdc42 activities are inhibitedwhile the cells are still attached to the substratum but the moreresponsive cells gets detached first. Incubation of MDA-MB-231 cellswith 500 nM MBQ-167 for 24 h resulted in ˜90% detachment of cells and aparallel decrease in Rac and Cdc42 activities, demonstrating that amajority of the cells were responsive to MBQ-167. Similarly theGFP-HER2-BM highly metastatic breast cancer cell line responded toMBQ-167 by inhibition of Rac and Cdc42 activities significantly in thedetached cell populations (FIGS. 10A, B). However, the non-metastaticmore epithelial MCF-7 cell line, which did not respond to MBQ-167 by thecell detachment phenotype, was also insensitive to MBQ-167 treatment inRac inhibition (Suppl. Table 2). This may be due to differences in theRac and Cdc42 GEFs that are expressed and activated in metastatic breastcancer cell lines (MDA-MB-231) compared to the less metastatic moreepithelial MCF-7 cell line.

Next, as a measure of the specificity of MBQ-167 as a Rac/Cdc42inhibitor, the IC₅₀s for Rac and Cdc42 activation were also determinedfollowing 24 h in MBQ-167 using combined attached and detachedpopulations. Results show that MBQ-167 inhibits Rac 1/2/3 activity inthe MDA-MB-231 cells with an IC₅₀ of 103 nM, and Cdc42 activity with anIC₅₀ of 78 nM (FIGS. 3D, E). Since the IC₅₀ for Rac inhibition byEHop-016 is 1.1 μM and Cdc42 inhibition is ˜8 μM (16), MBQ-167 is 10×more potent than EHop-016 for Rac inhibition and 100× more potent forCdc42 inhibition.

To indirectly determine the specificity of MBQ-167 for inhibiting Racactivation by GEFs, Rac activity was determined from previouslycharacterized MDA-MB-435Br cells expressing a control vector orconstitutively active (Rac1G12V). However, MBQ-167 did not affect theRac activity of this cell line expressing a Rac1(G12V) (FIG. 7),indicating that constitutive activation of Rac1 desensitizes the cellsto inhibition by MBQ-167. Moreover, as demonstrated from activationassays for Rac, Cdc42, and Rho from attached and detached cellpopulations, MBQ-167 did not affect the related GTPase Rho activation inboth cell populations of MDA-MB-231 and GFP-HER2-BM metastatic cancercells (Table 1, FIG. 10).

MBQ-167 Inhibits Rac and Cdc42 Downstream Effectors

To investigate the effect of MBQ-167 on Rac/Cdc42 signaling, its effecton the major Rac/Cdc42 downstream effector PAK was investigated. Thephosphorylation status of several PAK residues was analyzed by westernblotting, as a measure of its activity. At 250 nM, 24 h treatment withMBQ-167 inhibited PAK1 and PAK2 phosphorylation at the T423/T402 andS199/S192 residues in the detached population of MDA-MB-231 cells.Except for PAK1^(T423), phosphorylation of all of these residues wassignificantly decreased in the attached population as well. Even thoughPAK1^(T423) phosphorylation was not inhibited in the attached cells, thereduction in the homologous PAK2 phosphorylation sites indicates apreferential inhibition of PAK2 in the attached cells (FIGS. 4A, B).Interestingly, MBQ-167 induced a dramatic increase in thephosphorylation of the PAK1^(S144) (FIG. 4A). However, overall PAKactivity is inhibited by MBQ-167, because the activating phosphorylation(Y507/T508) of the direct PAK substrate LIM kinase (LIMK) and theinactivating phosphorylation (S3) of cofilin (actin depolymerizationfactor), a downstream effector of LIMK, were both decreased followingMBQ-167 treatment. The decrease in cofilin phosphorylation was evidentafter 12 h following 250 nM MBQ-167 (FIG. 4C), indicating activation ofcofilin, which account for the observed actin cytoskeletal restructuring(FIG. 2B). Moreover, in the MCF7 cell line, which did not respond toMBQ-167 by cell detachment or inhibition of Rac activation, MBQ-167 alsodid not affect PAK activity. Similarly, in the NCI-N87 human metastaticgastric cancer cell line, MBQ-167 inhibited the viability and theactivity of PAK isoforms PAK1 and PAK4, which have been implicated ingastric cancer malignancy (FIG. 8B).

Rac activity has also been shown to directly stimulate the activity ofthe transcription factor, STAT3 (30). As seen in FIG. 4D and FIG. 12A,MBQ-167 decreased STAT3 activity following 24 h exposure in both theattached and detached populations of MDA-MB-231 and GFP-HER2-BM cells.However, MBQ-167 did not affect mitogen activated protein kinase (MAPK)activities, either p38-MAPK or the p42/44 MAPK, as well as Aktactivities as demonstrated by western blotting with phospho-specificantibodies (FIG. 13).

MBQ-167 Affects Cancer Cell Polarity

Human breast cancer cells were visualized by bright field microscopyfollowing MBQ-167 treatment. At ≥100 nM, starting at six hours, MBQ-167induced a loss of polarity in metastatic breast cancer cells. Treatmentwith 500 nM MBQ-167 for 24 h resulted in ˜95% cell rounding anddetachment from the substratum in metastatic MDA-MB-231 cells (FIG. 2Aupper panel). Moreover, MBQ-167 induced this phenotype in multiplemesenchymal cancer cell types including GFP-HER2-BM, MDA-MB-468, andHs578t human breast cancer cells, as well as Mia-PaCa-2 pancreaticcancer cells, SKOV3 ovarian cancer cells, AGS and NCI-N87 gastric cancercells, and SH-SY5Y neuroblastoma cells (FIG. 9). On the other hand,non-cancer mammary epithelial MCF10A and epithelial breast cancer MCF-7cells were resistant to MBQ-167 and remained polarized and attached toeach other and the substratum (FIG. 2A, bottom panel). This indicatesthat MBQ-167 only affects cancer cell lines that have gone throughepithelial-mesenchymal (EMT) transition, and does not affect non-cancerepithelial cell lines or cancerous epithelial cell lines.

To further investigate the effect of MBQ-167 on MDA-MB-231 cells,immunofluorescence microscopy was performed following 0-500 nM MBQ-167to detect actin dynamics (by Rhodamine phalloidin) and focal adhesions(by anti-p-tyrosine and anti-vinculin). MBQ-167 rearranged the actincytoskeleton and focal adhesions to result in loss of cell polarity andattachment to the extracellular matrix (ECM), with a marked reduction inboth Rac-regulated lamellipodia/invadopodia and Cdc42-inducedmicrospikes and filopodia (FIG. II). Moreover, in MBQ-167-treated cells,the focal adhesions were reduced from the cell edge and rearranged fromthe cytoskeleton to the center of the rounded detaching cells.

Therefore, MBQ-167 inhibits cell polarization, extension of lamellipodiaand filopodia, and focal adhesion formation at the leading cell edge(FIG. 2B, C).

Growth of yeast cells that only express Cdc42 and not Rac were alsoinhibited by MBQ-167 with a characteteristic loss in bud polarity (FIG.15).

MBQ-167 Inhibits Cell Migration and Mammosphere Formation

Rac/Cdc42 and its downstream effector PAK directly regulate cellmigration. The effect of MBQ-167 on MDA-MB-231 cell migration wasinvestigated. The two cell populations (detached and attached) wererecovered following 18 h in MBQ-167 and equal numbers of cells(vehicle-treated, and MBQ-167-treated attached and detached populations)were used for a Transwell assay for 6 h. This short incubation time isnot sufficient for MDA-MB-231 cell division (doubling time of 38 h) orinhibition of cell viability (Suppl. FIG. 8B, C). Therefore, the assayonly measures the efficiency of cell migration.

MBQ-167 treatment reduced directed migration of the attached MDA-MB-231cell population by ˜60-70% at 250 and 500 nM. In the detachedpopulation, MBQ-167 (250 and 500 nM) inhibited cell migration by ˜90% ina statistically significant manner (FIG. 4E). In the more metastaticGFP-HER2-BM cell line, 250 and 500 nM MBQ-167 inhibited cell migrationby 80-90% in both attached and detached cells. These results wereconfirmed in a wound healing assay where 250 and 500 nM MBQ-167treatment for 24 h resulted in statistically significant ˜80 and ˜90%inhibition of wound closure, respectively (FIG. 4F). STAT3 and Racactivities have been implicated in enhanced breast cancer stem cell-likeproperties and therapy resistance. The capacity of MBQ-167 to targetcancer stem cell populations using a mammosphere formation assay.Addition of MBQ-167 once for four days reduced the mammosphere formingefficiency of MDA-MB-231 cells by ˜50% following 24 h treatment withMBQ-167 (FIG. 4F).

MBQ-167 Inhibits Cell Survival

MBQ-167 induces a phenotype characterized by cell rounding, loss oflamellipodia, and eventual detachment from the surface substratum (FIG.2). Therefore, we tested the potential of MBQ-167 to induce anoikis:apoptosis due to dissolution of integrin-mediated cell to ECMattachments. It should be emphasized that the metastatic cancer cellsthat detach in response to MBQ-167 following 24 h treatment are viable,as evidenced by trypan blue exclusion from live cells (Suppl. FIG. 8A).These detached cells also have the capacity for regrowth when replatedwithout MBQ-167 (data not shown). As shown in Supplemental FIG. 8B,MDA-MB-231 cells are 100% viable at concentrations ≤300 nM for 24 h. At24 h MBQ-167 treatment, ˜75% of MDA-MB-231, GFP-HER2-BM, and MCF-7breast cancer cells, as well as the MCF-10 mammary epithelial cells areviable even at 5 μM MBQ-167. Prolonged treatment for 48, 96, and 120 hwith MBQ-167 results in cell detachment from the substratum and loss ofcell viability.

FIG. 5A shows a MTT assay following MBQ-167 treatment for 120 h for themetastatic cancer cells MDA-MB-231 and GFP-HER2-BM and the non-cancermammary epithelial cells MCF10A, when we obtained ˜100% cell death forall cell types at high concentrations (1000 nM) of MBQ-167. This assayincludes both detached and attached cells in the case of the metastaticcancer cells. MBQ-167 at 120 h decreased the viability of MDA-MB-231 andGFP-HER2-BM cells with a GI₅₀ of 110 nM and 150 nM respectively.However, the GI₅₀ for the MCF10A epithelial cells at 350 nM MBQ-167 was˜3× higher (FIG. 5A). It should be noted that MBQ-167 inhibits Rac andCdc42 activities with IC₅₀s in the ˜100 nM range at 24 h when theMDA-MB-231, GFP-HER2-BM, MCF-7, and MCF-10 cells are still viable. Next,whether the effect of MBQ-167 on cell viability is due to cell cyclearrest by flow cytometry was determined. As shown in FIG. 5B, MBQ-167significantly arrested the cell cycle of MDA-MB-231 cells in the G2/Mphase.

To evaluate whether the cell cycle arrest was accompanied by an increasein apoptosis, the activity of the effector caspases3/7 in whole cellpopulations (both attached and detached). A dose-dependent increase wasobserved for caspase-3/7 activity in both MDA-MB-231 and GFP-HER2-BMcell lines after 24 h in MBQ-167 (FIG. 5C, left panel). To determinewhether MBQ-167 induces anoikis, the relative levels of caspase-3/7activities were analyzed in the attached and detached MDA-MB-231 cellpopulations following 24 h at 250 nM MBQ-167. There was significant˜15-fold increase in caspase-3/7 activity in the detached populationcompared to the attached population of MDA-MB-231 cells (FIG. 5C, rightpanel), the effect of MBQ-167 on apoptosis was validated by showingincreased Annexin V staining in MDA-MB-231 cells following 250 or 500 nMMBQ-167 (FIG. 14). In 500 nM MBQ-167, cells also demonstrated theclassic blebbing associated with apoptosis. Finally, to explore theeffect of MBQ-167 on mitochondrial apoptosis, the expression of thepro-survival proteins Bcl-2, Bcl-xL, and Mcl-1 was examined by westernblotting. A significant decrease was found in the expression ofpro-survival proteins after 24 h with 250 nM MBQ-167 (FIG. 5D).

MBQ-167 Inhibits Mammary Tumor Progression of HER2 Type Breast Cancer inImmunocompromised Mice

To test the effect of MB Q-167 on mammary tumor progression, nude micewere used to establish mammary fatpad tumors from GFP-HER2-BM cells. Oneweek following mammary tumor establishment, the mice were treated 3× aweek with 0, 1, or 10 mg/kg BW MBQ-167 by i.p. for 65 days. The vehicletreated mice demonstrated a linear increase in tumor growth, whileMBQ-167-treated mice demonstrated a statistically significant reductionin tumor growth (FIG. 6A). At sacrifice, 1.0 mg/kg BW of MBQ-167resulted in a ˜80% reduction in tumor growth, and the 10 mg/kg BWMBQ-167 treatment resulted in ˜95% reduction in tumor growth. SinceEHop-016 only exerts ˜40% reduction of tumor growth at 10 mg/kg BW,MBQ-167 is 10× more effective than EHop-016 (FIG. 6A).

The optimal % change in tumor size, which takes into account theindividual tumor growth for each treatment, showed that the tumors frommice treated with 1 mg/kg BW MBQ-167 demonstrated a 58% growth changecompared to controls (100%), while tumors from mice treated with 10mg/kg BW MBQ-167 demonstrated only a 9% increase in tumor size (Suppl.Table 3). These data indicate that even though there was no tumorregression during the time of study, there was a drastic reduction intumor growth in the 10 mg/kg BW MBQ-167 treated mice.

When the tumor growth delay was quantified, the control mice doubled in8 days, and the MBQ-167 treated mice demonstrated similar doubling timesfor both treatments (10 and 11 days). However, at the second doubling(2²), there was a delay in tumor growth of the MBQ-167 treated mice,where the tumors from control treated mice reached 2² in 14.5 days,while the tumors from 1 and 10 mg/kg BW treated mice were similar toeach other by reaching 2² in 30 days. By the 3^(rd) doubling, there wasalso a disparity between the two MBQ-167 treatments, where the tumorsfrom control mice reached 2³ in 27 days, the 1 mg/kg BW MBQ-167 treatedtumors took 57 days to reach the same size, and the 10 mg/kg BW treatedtumors never reached 2³ in tumor growth. Similarly, only the controltumors reached 2⁴ in 33 days, while the tumors from both MBQ-167 treated(1 and 10 mg/kg BW) mice never reached this size. This resultdemonstrates a significant inhibition in tumor growth initiated after 24days of MBQ-167 treatment (FIG. 6A). This drastic reduction in tumorgrowth following MBQ-167 treatment resulted in no metastases to all ofthe organs tested (FIG. 6B).

MBQ-167 Inhibits Mammary Tumor Progression of Triple Negative BreastCancer in Immunocompromised Mice. Mammary fat pad tumors wereestablished in severe combined immunodeficiency (SCID) mice using greenfluorescent protein (GFP) tagged-MDA-MB-231 breast cancer cells. Oneweek later, mice were treated with 0, 1.0, 5.0, or 10.0 mg/kg bodyweight (BW) MBQ-167 by intraperitoneal injections 3× a week for 50 days.Mammary tumor growth was quantified from GFP image analysis. Resultsshow average tumor growth in 50 days as a function of the size of eachtumor on day 01. Vehicle control (0 mg/kg BW MBQ-167) is represented as100% tumor growth. Administration of 1.0 mg/kg BW of MBQ-167 resulted ina 90% inhibition of tumor growth, 5.0 mg/kg BW treatment resulted in a95% inhibition of tumor growth and the 10 mg/kg BW MBQ-167 treatmentresulted in 100% inhibition of tumor growth in 50 days (FIG. 16).

MBQ-167 is not Toxic to Immunocompromised Mice

The mice from this study were also examined once a week for potentialtoxicity. The mice treated with vehicle or MBQ-167 did not show anysignificant weight loss or phenotypic changes during the 65-day study(FIG. 6C). At necropsy, livers were harvested, lysed and subjected toliver enzyme assays as a test for potential toxic effects. FIG. 6D showsthat MBQ-167 does not affect ALP activity in the livers of MBQ-167treated nude mice (1 and 10 mg/kg BW). However, the liver ALT levelswere significantly increased by 10 mg/kg BW MBQ-167 treatment,indicating a potential metabolism of MBQ-167 at higher concentrations(FIG. 6E).

Molecules which function as inhibitors of the Ras-homologous (Rho)family of small GTPases (e.g. Rac and Cdc42) and their use to treatcancers including breast, pancreatic, ovarian, prostate, gastric, andneuroblastoma, where these GTPases are overexpressed or hyperactivated,and diseases where activation of Rho GTPases plays a pivotal role aremediated through these proteins.

A compound according to general formula I

Wherein:

R1 is hydrogen, C1-6 alkyl, or C3-6 cycloalkyl.

R² is independently in each occurrence hydrogen, halide, hydroxyl,cyano, C₁₋₆ alkyl, C₃₋₆ cycloalkyl, C₁₋₆ haloalkyl, nitro, amino, etc.

R³ is aryl, heteroaryl, indol-5-yl, benzimidazol-5-yl, indazol-5-yl,C₁₋₆ alkyl, C₂₋₆.

hydroxyalkyl, C3-6 cycloalkyl, or R^(a),R^(b)N[CR⁴]2-6.

R^(a) and R^(b) are independently hydrogen, C₁₋₆ alkyl, or C₃₋₆cycloalkyl

R⁴ is independently in each occurrence hydrogen or C₁₋₆ alkyl.

A and B are independently hydrogen, halide, hydroxyl, cyano, C₁₋₆ alkyl,C₃₋₆ cycloalkyl, C₁₋₆ haloalkyl, nitro, amino, etc.

In the compound of general formula 1, where A and B form a ring system:

Wherein:

R5 is independently in each occurrence hydrogen, halide, hydroxyl,cyano, C1-6 alkyl, C3-6 cycloalkyl, C1-6 haloalkyl, nitro, amino.

A method of inhibiting Rac and/or Cdc42 in a cell includes contactingthe cell with a compound of general formula 1.

A method of treating a patient with cancer or hyperproliferativedisorders is by administering to the patient a compound of generalformula 1.

The cancer or hyperproliferative disorders include breast cancer,prostate cancer, neuroblastoma, ovarian cancer, pancreatic cancer, orgastric cancer.

The biochemical characterization of MBQ-167, which inhibits Rac andCdc42 activation. Rac and Cdc42 are pivotal signaling intermediateswhose dysregulation has been implicated in oncogenic transformation,cancer progression, metastasis, and multiple diseases. Recent studies,including the inventors' own, have shown that targeting Rac and Cdc42has potential for metastatic cancer therapy. However, the current smallmolecule inhibitors of Rac and Cdc42 are only effective at micromolarconcentrations.

To develop a new, more potent inhibitor of Rac and Cdc42 with anIC₅₀<1.0 μM, the novel derivative MBQ-167 shows improved efficacy inmetastatic breast cancer cells by inhibiting Rac activity with an IC₅₀of ˜103 nM, and Cdc42 with an IC₅₀ of 78 nM. This concentrationdependent response of breast cancer cells to MB Q-167 demonstrates thatMBQ-167 is specifically inhibiting the biochemical activation of the Racand Cdc42. However, MBQ-167 did not inhibit the Rac activity of cellsexpressing dominant active Rac1(G12V) indicating that MBQ-167 isspecifically inhibiting Rac1 activation. Moreover, our data show thatMBQ-167 does not affect the activation of the related GTPase Rho.Therefore, we expect this new molecule to be useful as a tool forprobing Rac and Cdc42 function in responsive cell types.

MBQ-167 was an effective inhibitor of the Rac and Cdc42 downstreameffector PAK. Interestingly, MBQ-167 induced an increasedautophosphorylation of S144 (PAK1), the activation of which is notessential, but contributes to the activity of the PAK kinase domain.This result may be due to a feedback mechanism compensating forRac/Cdc42 inhibition. Nevertheless, phosphorylations in the PAK kinasedomains, as well as the PAK effectors LIMK and cofilin, a potentregulator of actin filament dynamics during cell migration, weresignificantly inhibited by MBQ-167. Therefore, we conclude that overall,MBQ-167 inhibits PAK activity, contributing to a reduction in actincytoskeletal extensions and cell migration. Since Rac and Cdc42 alsoregulate Wiskott Aldrich Syndrome protein (WASP)-family members thatcontribute to actin dynamics, MBQ-167 may exert additional inhibitoryeffects on the cytoskeleton.

Furthermore, Cdc42 regulates cell polarity through the polarity proteinportioning defective proteins (PARE, 3), which stabilize microtubulesduring directed migration. Using a haploid derivative of the yeaststrain BY4741, where the essential gene Cdc42 was knocked outconditionally via a tetracycline inducible promoter, MBQ-167 exerts asimilar phenotype to the cells with reduced Cdc42 expression. Cdc42knockdown abolishes cell polarity where the yeast buds (daughter cells)are not aligned symmetrically with the mother cells. A similar non-polareffect was also observed on yeast cell budding in the presence ofMBQ-167. This mutant phenotype was more pronounced in the yeast cellswith both Cdc42 knockdown and MBQ-167 treatment demonstrating thatMBQ-167 may inhibit the highly conserved yeast Cdc42 to regulate cellpolarity. As expected, MBQ-167 treatment also enhanced the growthinhibitory effects of Cdc42 knockdown.

The regulation of microtubule dynamics by Rac and Cdc42 activities arealso critical for cell cycle progression, where Cdc42, and thus PAK,controls mitotic spindle formation and cell cycle progression in G₂/M(41, 42). Therefore, the observed MBQ-167-mediated metastatic breastcancer cell cycle arrest in the G₂/M phase may be a consequence ofRac/Cdc42/PAK inhibition by MB Q-167.

The decreased Rac/Cdc42/PAK activities, cell viability, loss of cellpolarity, and detachment from the substratum in response to MBQ-167 islimited to cancer cells that have undergone epithelial to mesenchymaltransition (EMT) but not to epithelial cancer or non-cancer cells. Thisselective response to MBQ-167 may be due to the differential expressionand activities of Rac and Cdc42 GEFs in different breast cancer celllines, where only a subset of the ˜80 known Rac and Cdc42 GEFs areexpected to be expressed and activated in the metastatic breast cancercell lines that were investigated. Moreover, the currently availableRac/Cdc42 inhibitors also inhibit only a subset of Rac/Cdc42 GEFs. Forinstance NSC23766 inhibits only Tiam-1/and Trio activation of Rac, whileEHop-016 is a specific inhibitor of the Vav/Rac interaction. Therefore,MBQ-167 may inhibit only a subset of Rac/Cdc42 GEFs that arepreferentially expressed/activated in the more metastaticmesenchymal-like cancer cells lines.

Additionally, the relative insensitivity of epithelial-like cells toMBQ-167 may be because the hemidesmosomes in epithelial cells areprimarily regulated by α6β4-integrin mediated attachments to theintermediate filament cytoskeleton, which are not directly regulated byRac and Cdc42. In contrast, the focal adhesions in mesenchymal cells,which are regulated by multiple integrin sub units to form attachmentswith the actin cytoskeleton, are under Rac/Cdc42/PAK regulation.Therefore, the observed reduction and reorganization of focal adhesionsin MBQ-167 treated cancer cells may reflect inhibition of theRac/Cdc42/PAK regulated integrin-mediated focal adhesion assembly at thecell leading edge.

Focal adhesions are not only important for directed migration,disruptions in proper regulation of cell adhesion to the ECM can resultin anoikis, apoptosis induced by inadequate or inappropriate cell-matrixinteractions. In this context, Rac1 has been shown to confer anoikisresistance. Data disclosed with caspase assays and reduction inpro-survival BCl2-homology proteins validate the hypothesis that MBQ-167acts as an anticancer agent by inducing anoikis. Data show that only thedetached breast cancer cells respond to MBQ-167 increased caspase3/7activities, indicating that cell detachment precedes apoptosissignaling, as would be predicted during anoikis. Moreover, we show thatMBQ-167 selectively decreases the viability of cancer cell lines thathave undergone EMT, without affecting the non-cancer cell line MCF10A.This cell line specificity could be due to differences in the dependenceon Rac/Cdc42/PAK signaling and the accompanying integrin engagement andfocal adhesion assembly, in the more migratory mesenchymal cellscompared to the epithelial cells. In addition, MBQ-167 may have similareffects in multiple other cancers, including a number of ovarian,gastric, pancreatic, and neuroblastoma cell lines that have undergoneEMT. Since EMT is associated with more stem cell-like properties,therapy resistance, and disease recurrence, MBQ-167 has potential toreduce therapy resistance. Moreover, the fact that MBQ-167 is effectiveagainst the KRAS mutant MIA PaCa-2 cell line demonstrates its ability totarget oncogenic RAS dependent cancers.

MBQ-167 also inhibits STAT3 phosphorylation, a Rac-regulatedtranscription factor shown to be active in several cancers. Since STAT3activity increases the expression of several genes involved in cellcycle progression, its decrease in activity may contribute to theobserved cell cycle arrest by MBQ-167 treatment. Importantly, STAT3transcriptionally regulates all three of the pro-survival BCl-2 familygenes analyzed in this study. Furthermore, several reports show thatcancer stem cell like properties are dependent on STAT3 activity.Accordingly, MBQ-167 decreases the mammosphere forming efficiency ofMDA-MB-231 cells by ˜50%. These results suggest that MBQ-167 may befurther effective as an anti-cancer therapeutic by targeting cancer stemcell like populations, perhaps specifically inhibits cancer stem cellactivity.

Finally, MBQ-167 reduces mammary fat pad tumor size starting as early as3 wks following treatment, with a 91% reduction by 2 months at anon-toxic concentration of 10 mg/kg BW. The drastic reduction in mammarytumors also resulted in a 100% inhibition of metastases to all organstested, probably because less cells were shed by the small tumors. Asevidenced by the in vitro data, the reduced tumor size in response toMBQ-167 treatment is predicted to be due to inhibition of Rac/Cdc42/PAKsignaling ultimately leading to a loss in cell viability, growth, andpolarity causing the cells to detach from the tumor and undergo anoikis.Since we did not observe any metastases in mice treated with MBQ-167,any cells detached from the primary tumor probably go through anoikis,and do not survive in the circulation.

Similarly, in severe combined immunodeficiency (SCID) mice withMDA-MB-231 mammary tumors, MBQ-167 inhibited tumor growth by 90%, 95%,and 100% at doses of 1, 5, and 10 mg/kg BW, respectively (FIG. 16).

Taken together, MBQ-167 is an effective Cdc42 and Rac inhibitor thatsignificantly decreases downstream signaling and cancer promoting cellfunctions to ultimately reduce mammary tumor growth with 10× morepotency than the first described Rac inhibitor EHop-016. However, theeffects of MBQ-167 on the metastatic cancer cell phenotype, where thecells detach from the substratum to ultimately undergo apoptosis byanoikis mechanisms, may be due to additional effects of MBQ-167 onintegrin signaling or alternate mechanisms. Nevertheless, the dramaticeffect of MBQ-167 on mouse mammary tumor growth warrants furtherdevelopment of MBQ-167 as an anticancer therapeutic.

EXAMPLES

MBQ-167 (9-Ethyl-3-(5-phenyl-[1,2,3]triazol-1-yl)-9H-carbazole) Data

FIG. 1. Freshly prepared 3-azido-9-ethylcarbazole was reacted withmagnesium phenylacetylide to generate the 4-halomagnesiotriazoleintermediate, that was quenched with 10% ammonium chloride to furnish9-Ethyl-3-(5-phenyl-[1,2,3]triazol-1-yl)-9H-carbazole (MBQ-167) in 86%yield.

FIG. 2A; FIG. 2B. MBQ-167 affects metastatic cancer cell polaritywithout affecting non-metastatic cancer cells. At ≥100 nM, starting atsix hours MBQ-167 induces a loss of polarity and detachment from thesubstratum in MDA-MB-231 metastatic breast cancer cells but not in MCF-7non-metastatic human breast cancer cells. 250 nM MBQ-167 results in ˜70%of the cells losing polarity and assuming a rounded phenotype. Higherconcentrations of MBQ-167, i.e. 500 nM for 24 h results in a ˜90% lossin cell polarity and detaches from the substratum.

FIG. 2A; FIG. 9. This dramatic rounding and detachment in response toMBQ-167 is demonstrated by HER2 type and triple negative breast cancer(TNBC) mesenchymal breast cancer cells (MDA-MB-231, Hs578t, MDA-MB-468,and HER2-BM), as well as other metastatic cancer cell lines: Mia-PaCa-2pancreatic cancer (with G12C kRas mutation), SKOV3 ovarian cancer,NCI-N87 gastric cancer, and SH-SY5Y neuroblastoma. This effect was lessin the more epithelial MCF-10A and MCF-7 breast cancer cells and AGSprimary gastric cancer cells.

FIG. 15. In Saccharomyces cerevisiae (budding yeast) that only expressesCdc42, which regulates polarity and cell division, treatment with 100microM MBQ-167 inhibited yeast bud polarity and growth, suggesting thatthe effect on cell polarity may be via inhibition of Cdc42 by MBQ-167.

FIG. 2B. Staining for F-actin by Rhodamine phalloidin shows a loss ofinvadopodia and filopodia, F-actin based motile structures regulated byRac and Cdc42, in response to MBQ-167.

FIGS. 2B, C. Immunostaining with an anti phosphotyrosine or vinculinantibody, for focal adhesions that are formed by the engagement ofintegrin receptors with the ECM, demonstrate a dramatic rearrangement offocal adhesions during cell rounding, where the focal adhesions movedfrom the cell edge to the cell center and appear more disorganized.

FIG. 3. MBQ-167 inhibits Rac 1/2/3 activity in the MDA-MB-231 humanmetastatic breast cancer cell line with an IC50 of 103 nM, and Cdc42activity with an IC50 of 78 nM.

Table 1. At 250 nM MBQ-167 inhibts the Rac and Cdc42 activities ofMDA-MB-231 cells without affecting the activity of the close isoformRho.

FIG. 10. MBQ-167 also inhibits the Rac and Cdc42 activities ofGFP-HER2-BM human metastatic breast cancer cells. MBQ-167 does notaffect the activity of Rho.

Table 2. MBQ-167 (250 nM) does not affect the Rac activity of MCF-7, anon-metastatic human breast cancer cell line.

FIG. 7. MBQ-167 also does not inhibit the Rac activity of aconstitutively active metastatic cancer cell line (MDA-MB-435-Rac1G12V)indicating that MBQ-167 is inhibiting the loading of GTP to Rac.

FIG. 3, FIG. 10. In MDA-MB-231 and GFP-HER2-BM metastatic breast cancercells, Rac, Cdc42, and the Rac and Cdc42 downstream effectorp21-activated kinase PAK activities were reduced by 75, 85, and 90%respectively, in the detached cells compared to vehicle controls, whilethese activities were less reduced (˜25%) in the attached population.

FIG. 8. In NCI-N87 gastric cancer cells, MBQ-167 significantly decreasesthe activity (but not expression) of the Rac and Cdc42 downstreameffector p21-activated kinase (PAK 1, 2, 4), as demonstrated by westernblotting with antibodies to total and active phospho residues.

FIG. 11. MBQ-167 does not inhibit PAK activities of MCF-7 primary breastcancer cell line.

FIGS. 4B, 4C. In MDA-MB-231 cells, 250 nM MBQ-167 inhibits downstreamPAK signaling as evidenced by western blotting for the activatingphosphorylation (Y507/T508) of the direct PAK substrate LIM kinase(LIMK) and the inactivating phosphorylation (S3) of the LIMK substratecofilin (actin depolymerization factor).

FIG. 4D, FIG. 12A. In MDA-MB-231 and GFP-HER2-BM cells, MBQ-167 reducesthe activity of signal transducer and activator of transcription(STAT3). However, MBQ-167 does not affect the p42/44 mitogen activatedkinase (MAPK), p38-MAPK, or Akt activities of MDA-MB-231 cells.

FIG. 4E, 4F; FIG. 12B. MBQ-167 mediated inhibition of Rac/Cdc42signaling results in 80-90% inhibition of MDA-MB-231 and GFP-HER2-BMcell migration as shown by Transwell and wound healing assays.

FIG. 4G. The mammosphere forming efficiency of MDA-MB-231 cells issignificantly reduced by ˜50%, following a 4-day treatment with MBQ-167.

FIG. 5A. MDA-MB-231 and GFP-HER2-BM metastatic breast cancer cellsincubated in 1000 nM MBQ-167 for 120 h undergoes ˜100% cell death with aGI₅₀ of 110 nM for MDA-MB-231 cells and a GI₅₀ of 150 nM for GFP-HER2-BMcells. MBQ-167 also does not affect the viability of MCF7 non-metastaticbreast cancer cells or MCF-10A mammary epithelial cells at theseconcentrations. The GI₅₀ for the MCF10A epithelial cells at 350 nMMBQ-167 is ˜3× higher than that for the metastatic breast cancer cells.

FIG. 8A. NCI-N87 viability. 250 nM MBQ-167 reduced the viability ofNCI-N87 cells by 55%, as analyzed from a MTT assay.

FIG. 5B. MBQ-167 treatment for 24 h to combined attached and detachedMDA-MB-231 cells results in a G₂/M phase arrest.

FIG. 5C; FIG. 14. The detached MDA-MB-231 breast cancer cells respond toMBQ-167 by increased caspase 3/7 activities. The attached cells initiateapoptosis in response to 250-500 nM MBQ-167 at 6 h as shown by Annexin Vstaining.

FIG. 5D. MBQ-167 treatment of MDA-MB-231 cells for 24 h results inreduced expression of the mitochondrial pro-survival proteins of theBcl-2 family (Bcl-2, Bcl-XL, and MCl-1).

FIG. 6A, 6B. Tables 3, 4. In nude mice with mammary fat pad tumorsestablished with GFP-HER2-BM cells, 1.0 mg/kg BW of MBQ-167 results in a˜80% reduction in tumor growth, and the 10 mg/kg BW MBQ-167 treatmentresults in ˜95% reduction in tumor growth in 65 days. Data indicates adrastic reduction in tumor growth in the 10 mg/kg BW MBQ-167 treatedmice, with a significant inhibition in tumor growth initiated after 24days of 1 or 10 mg/kg BW MBQ-167 treatment.

FIG. 16. MB-231 Mammary Tumor Growth. In severe combinedimmunodeficiency (SCID) mice bearing GFP-MDA-MB-231 mammary fat padtumors, 1.0 mg/kg BW of MBQ-167 resulted in a 90% inhibition of tumorgrowth, 5.0 mg/kg BW treatment resulted in a 95% inhibition of tumorgrowth and the 10 mg/kg BW MBQ-167 treatment resulted in 100% inhibitionof tumor growth in 50 days.

FIG. 6B. Due to this drastic reduction in tumor growth, MBQ-167 inhibitsmetastasis, to all of the organs tested (lungs, bone, heart, spleen,kidney, livers), by 100%.

FIG. 6C. MBQ-167 does not exert toxic effects on weight or phenotype ofnude or SCID immunocompromised mice.

FIG. 6D, 6E. MBQ-167 does not significantly affect liver alkalinephosphatase (ALP) activity but increases liver alanine transaminase(ALT) activity.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this disclosure belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entireties. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in a patent, application, or other publication thatis herein incorporated by reference, the definition set forth in thissection prevails over the definition incorporated herein by reference.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. It is further noted that the claims may be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

As used herein, the terms “including,” “containing,” and “comprising”are used in their open, non-limiting sense.

To provide a more concise description, some of the quantitativeexpressions given herein are not qualified with the term “about”. It isunderstood that, whether the term “about” is used explicitly or not,every quantity given herein is meant to refer to the actual given value,and it is also meant to refer to the approximation to such given valuethat would reasonably be inferred based on the ordinary skill in theart, including equivalents and approximations due to the experimentaland/or measurement conditions for such given value. Whenever a yield isgiven as a percentage, such yield refers to a mass of the entity forwhich the yield is given with respect to the maximum amount of the sameentity that could be obtained under the particular stoichiometricconditions. Concentrations that are given as percentages refer to massratios, unless indicated differently.

Except as otherwise noted, the methods and techniques of the presentembodiments are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See, e.g., Loudon, Organic Chemistry, Fourth Edition, NewYork: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith andMarch, March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Fifth Edition, Wiley-Interscience, 2001.

Chemical nomenclature for compounds described herein has generally beenderived using the commercially-available ACD/Name 2014 (ACD/Labs) orChemBioDraw Ultra 13.0 (Perkin Elmer).

It is appreciated that certain features of the disclosure, which are,for clarity, described in the context of separate embodiments, may alsobe provided in combination in a single embodiment. Conversely, variousfeatures of the disclosure, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination. All combinations of the embodimentspertaining to the chemical groups represented by the variables arespecifically embraced by the present disclosure and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace compounds thatare stable compounds (i.e., compounds that can be isolated,characterized, and tested for biological activity). In addition, allsubcombinations of the chemical groups listed in the embodimentsdescribing such variables are also specifically embraced by the presentdisclosure and are disclosed herein just as if each and every suchsub-combination of chemical groups was individually and explicitlydisclosed herein.

Definitions

As used herein, the term “alkyl” includes a chain of carbon atoms, whichis optionally branched and contains from 1 to 20 carbon atoms. It is tobe further understood that in certain embodiments, alkyl may beadvantageously of limited length, including C₁-C₁₂, C₁-C₁₀, C₁-C₉,C₁-C₈, C₁-C₇, C₁-C₆, and C₁-C₄, Illustratively, such particularlylimited length alkyl groups, including C₁-C₈, C₁-C₇, C₁-C₆, and C₁-C₄,and the like may be referred to as “lower alkyl.” Illustrative alkylgroups include, but are not limited to, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl,3-pentyl, neopentyl, hexyl, heptyl, octyl, and the like. Alkyl may besubstituted or unsubstituted. Typical substituent groups includecycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,mercapto, alkylthio, arylthio, cyano, halo, carbonyl, oxo, (═O),thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, C-carboxy, O-carboxy, nitro, and amino, or asdescribed in the various embodiments provided herein. It will beunderstood that “alkyl” may be combined with other groups, such as thoseprovided above, to form a functionalized alkyl. By way of example, thecombination of an “alkyl” group, as described herein, with a “carboxy”group may be referred to as a “carboxyalkyl” group. Other non-limitingexamples include hydroxyalkyl, aminoalkyl, and the like.

As used herein, the term “alkenyl” includes a chain of carbon atoms,which is optionally branched, and contains from 2 to 20 carbon atoms,and also includes at least one carbon-carbon double bond (i.e. C═C). Itwill be understood that in certain embodiments, alkenyl may beadvantageously of limited length, including C₂-C₁₂, C₂-C₉, C₂-C₈, C₂-C₇,C₂-C₆, and C₂-C₄. Illustratively, such particularly limited lengthalkenyl groups, including C₂-C₈, C₂-C₇, C₂-C₆, and C₂-C₄ may be referredto as lower alkenyl. Alkenyl may be unsubstituted, or substituted asdescribed for alkyl or as described in the various embodiments providedherein. Illustrative alkenyl groups include, but are not limited to,ethenyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, and the like.

As used herein, the term “alkynyl” includes a chain of carbon atoms,which is optionally branched, and contains from 2 to 20 carbon atoms,and also includes at least one carbon-carbon triple bond (i.e. C≡C). Itwill be understood that in certain embodiments, alkynyl may each beadvantageously of limited length, including C₂-C₁₂, C₂-C₉, C₂-C₈, C₂-C₇,C₂-C₆, and C₂-C₄. Illustratively, such particularly limited lengthalkynyl groups, including C₂-C₈, C₂-C₇, C₂-C₆, and C₂-C₄ may be referredto as lower alkynyl. Alkenyl may be unsubstituted, or substituted asdescribed for alkyl or as described in the various embodiments providedherein. Illustrative alkenyl groups include, but are not limited to,ethynyl, 1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl, and the like.

As used herein, the term “aryl” refers to an all-carbon monocyclic orfused-ring polycyclic groups of 6 to 12 carbon atoms having a completelyconjugated pi-electron system. It will be understood that in certainembodiments, aryl may be advantageously of limited size such as C₆-C₁₀aryl. Illustrative aryl groups include, but are not limited to, phenyl,naphthylenyl and anthracenyl. The aryl group may be unsubstituted, orsubstituted as described for alkyl or as described in the variousembodiments provided herein.

As used herein, the term “cycloalkyl” refers to a 3 to 15 memberall-carbon monocyclic ring, including an all-carbon 5-member/6-member or6-member/6-member fused bicyclic ring, or a multicyclic fused ring (a“fused” ring system means that each ring in the system shares anadjacent pair of carbon atoms with each other ring in the system) group,where one or more of the rings may contain one or more double bonds butthe cycloalkyl does not contain a completely conjugated pi-electronsystem. It will be understood that in certain embodiments, cycloalkylmay be advantageously of limited size such as C₃-C₁₃, C₃-C₉, C₃-C₆ andC₄-C₆. Cycloalkyl may be unsubstituted, or substituted as described foralkyl or as described in the various embodiments provided herein.Illustrative cycloalkyl groups include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclopentadienyl,cyclohexyl, cyclohexenyl, cycloheptyl, adamantyl, norbornyl,norbornenyl, 9H-fluoren-9-yl, and the like. Illustrative examples ofcycloalkyl groups shown in graphical representations include thefollowing entities, in the form of properly bonded moieties:

As used herein, the term “heterocycloalkyl” refers to a monocyclic orfused ring group having in the ring(s) from 3 to 12 ring atoms, in whichat least one ring atom is a heteroatom, such as nitrogen, oxygen orsulfur, the remaining ring atoms being carbon atoms. Heterocycloalkylmay optionally contain 1, 2, 3 or 4 heteroatoms. Heterocycloalkyl mayalso have one of more double bonds, including double bonds to nitrogen(e.g. C═N or N═N) but does not contain a completely conjugatedpi-electron system. It will be understood that in certain embodiments,heterocycloalkyl may be advantageously of limited size such as 3- to7-membered heterocycloalkyl, 5- to 7-membered heterocycloalkyl, and thelike. Heterocycloalkyl may be unsubstituted, or substituted as describedfor alkyl or as described in the various embodiments provided herein.Illustrative heterocycloalkyl groups include, but are not limited to,oxiranyl, thianaryl, azetidinyl, oxetanyl, tetrahydrofuranyl,pyrrolidinyl, tetrahydropyranyl, piperidinyl, 1,4-dioxanyl, morpholinyl,1,4-dithianyl, piperazinyl, oxepanyl, 3,4-dihydro-2H-pyranyl,5,6-dihydro-2H-pyranyl, 2H-pyranyl, 1, 2, 3, 4-tetrahydropyridinyl, andthe like. Illustrative examples of heterocycloalkyl groups shown ingraphical representations include the following entities, in the form ofproperly bonded moieties:

As used herein, the term “heteroaryl” refers to a monocyclic or fusedring group of 5 to 12 ring atoms containing one, two, three or four ringheteroatoms selected from nitrogen, oxygen and sulfur, the remainingring atoms being carbon atoms, and also having a completely conjugatedpi-electron system. It will be understood that in certain embodiments,heteroaryl may be advantageously of limited size such as 3- to7-membered heteroaryl, 5- to 7-membered heteroaryl, and the like.Heteroaryl may be unsubstituted, or substituted as described for alkylor as described in the various embodiments provided herein. Illustrativeheteroaryl groups include, but are not limited to, pyrrolyl, furanyl,thiophenyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridinyl,pyrimidinyl, quinolinyl, isoquinolinyl, purinyl, tetrazolyl, triazinyl,pyrazinyl, tetrazinyl, quinazolinyl, quinoxalinyl, thienyl, isoxazolyl,isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl,benzoxazolyl, benzthiazolyl, benzisoxazolyl, benzisothiazolyl andcarbazoloyl, and the like. Illustrative examples of heteroaryl groupsshown in graphical representations, include the following entities, inthe form of properly bonded moieties:

As used herein, “hydroxy” or ““hydroxyl” refers to an —OH group.

As used herein, “alkoxy” refers to both an —O-(alkyl) or an—O-(unsubstituted cycloalkyl) group. Representative examples include,but are not limited to, methoxy, ethoxy, propoxy, butoxy,cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and thelike.

As used herein, “aryloxy” refers to an —O-aryl or an —O-heteroarylgroup. Representative examples include, but are not limited to, phenoxy,pyridinyloxy, furanyloxy, thienyloxy, pyrimidinyloxy, pyrazinyloxy, andthe like, and the like.

As used herein, “mercapto” refers to an —SH group.

As used herein, “alkylthio” refers to an —S-(alkyl) or an—S-(unsubstituted cycloalkyl) group. Representative examples include,but are not limited to, methylthio, ethylthio, propylthio, butylthio,cyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio, andthe like.

As used herein, “arylthio” refers to an —S-aryl or an —S-heteroarylgroup. Representative examples include, but are not limited to,phenylthio, pyridinylthio, furanylthio, thienylthio, pyrimidinylthio,and the like.

As used herein, “halo” or “halogen” refers to fluorine, chlorine,bromine or iodine.

As used herein, “cyano” refers to a —CN group.

The term “oxo” represents a carbonyl oxygen. For example, a cyclopentylsubstituted with oxo is cyclopentanone.

As used herein, “bond” refers to a covalent bond.

The term “substituted” means that the specified group or moiety bearsone or more substituents. The term “unsubstituted” means that thespecified group bears no substituents. Where the term “substituted” isused to describe a structural system, the substitution is meant to occurat any valency-allowed position on the system. In some embodiments,“substituted” means that the specified group or moiety bears one, two,or three substituents. In other embodiments, “substituted” means thatthe specified group or moiety bears one or two substituents. In stillother embodiments, “substituted” means the specified group or moietybears one substituent.

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance may but need not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. For example, “wherein each hydrogenatom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3-to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, or mono- or bicyclicheteroaryl is independently optionally substituted by C₁-C₆ alkyl” meansthat an alkyl may be but need not be present on any of the C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to 7-memberedheterocycloalkyl, C₆-C₁₀ aryl, or mono- or bicyclic heteroaryl byreplacement of a hydrogen atom for each alkyl group, and the descriptionincludes situations where the C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, ormono- or bicyclic heteroaryl is substituted with an alkyl group andsituations where the C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, or mono- orbicyclic heteroaryl is not substituted with the alkyl group.

As used herein, “independently” means that the subsequently describedevent or circumstance is to be read on its own relative to other similarevents or circumstances. For example, in a circumstance where severalequivalent hydrogen groups are optionally substituted by another groupdescribed in the circumstance, the use of “independently optionally”means that each instance of a hydrogen atom on the group may besubstituted by another group, where the groups replacing each of thehydrogen atoms may be the same or different. Or for example, wheremultiple groups exist all of which can be selected from a set ofpossibilities, the use of “independently” means that each of the groupscan be selected from the set of possibilities separate from any othergroup, and the groups selected in the circumstance may be the same ordifferent.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which counter ions which may be used in pharmaceuticals.See, generally, S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm.Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts arethose that are pharmacologically effective and suitable for contact withthe tissues of subjects without undue toxicity, irritation, or allergicresponse. A compound described herein may possess a sufficiently acidicgroup, a sufficiently basic group, both types of functional groups, ormore than one of each type, and accordingly react with a number ofinorganic or organic bases, and inorganic and organic acids, to form apharmaceutically acceptable salt. Such salts include:

(1) acid addition salts, which can be obtained by reaction of the freebase of the parent compound with inorganic acids such as hydrochloricacid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, andperchloric acid and the like, or with organic acids such as acetic acid,oxalic acid, (D) or (L) malic acid, maleic acid, methane sulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaricacid, citric acid, succinic acid or malonic acid and the like; or

(2) salts formed when an acidic proton present in the parent compoundeither is replaced by a metal ion, e.g., an alkali metal ion, analkaline earth ion, or an aluminum ion; or coordinates with an organicbase such as ethanolamine, diethanolamine, triethanolamine,trimethamine, N-methylglucamine, and the like.

Pharmaceutically acceptable salts are well known to those skilled in theart, and any such pharmaceutically acceptable salt may be contemplatedin connection with the embodiments described herein. Examples ofpharmaceutically acceptable salts include sulfates, pyrosulfates,bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates,dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides,bromides, iodides, acetates, propionates, decanoates, caprylates,acrylates, formates, isobutyrates, caproates, heptanoates, propiolates,oxalates, malonates, succinates, suberates, sebacates, fumarates,maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates,chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates,methoxybenzoates, phthalates, sulfonates, methylsulfonates,propylsulfonates, besylates, xylenesulfonates, naphthalene-1-sulfonates,naphthalene-2-sulfonates, phenylacetates, phenylpropionates,phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates,tartrates, and mandelates. Lists of other suitable pharmaceuticallyacceptable salts are found in Remington's Pharmaceutical Sciences, 17thEdition, Mack Publishing Company, Easton, Pa., 1985.

For a that contains a basic nitrogen, a pharmaceutically acceptable saltmay be prepared by any suitable method available in the art, forexample, treatment of the free base with an inorganic acid, such ashydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid,nitric acid, boric acid, phosphoric acid, and the like, or with anorganic acid, such as acetic acid, phenylacetic acid, propionic acid,stearic acid, lactic acid, ascorbic acid, maleic acid, hydroxymaleicacid, isethionic acid, succinic acid, valeric acid, fumaric acid,malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid,oleic acid, palmitic acid, lauric acid, a pyranosidyl acid, such asglucuronic acid or galacturonic acid, an alpha-hydroxy acid, such asmandelic acid, citric acid, or tartaric acid, an amino acid, such asaspartic acid or glutamic acid, an aromatic acid, such as benzoic acid,2-acetoxybenzoic acid, naphthoic acid, or cinnamic acid, a sulfonicacid, such as laurylsulfonic acid, p-toluenesulfonic acid,methanesulfonic acid, or ethanesulfonic acid, or any compatible mixtureof acids such as those given as examples herein, and any other acid andmixture thereof that are regarded as equivalents or acceptablesubstitutes in light of the ordinary level of skill in this technology.

The disclosure also relates to pharmaceutically acceptable prodrugs ofthe compounds and treatment methods employing such pharmaceuticallyacceptable prodrugs. The term “prodrug” means a precursor of adesignated compound that, following administration to a subject, yieldsthe compound in vivo via a chemical or physiological process such assolvolysis or enzymatic cleavage, or under physiological conditions(e.g., a prodrug on being brought to physiological pH is converted tothe compound in accordance with the present disclosure). A“pharmaceutically acceptable prodrug” is a prodrug that is non-toxic,biologically tolerable, and otherwise biologically suitable foradministration to the subject. Illustrative procedures for the selectionand preparation of suitable prodrug derivatives are described, forexample, in “Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985.

Any formula depicted herein is intended to represent a compound of thatstructural formula as well as certain variations or forms. For example,a formula given herein is intended to include a racemic form, or one ormore enantiomeric, diastereomeric, or geometric isomers, or a mixturethereof. Additionally, any formula given herein is intended to referalso to a hydrate, solvate, or polymorph of such a compound, or amixture thereof. For example, it will be appreciated that compoundsdepicted by a structural formula containing the symbol “

” include both stereoisomers for the carbon atom to which the symbol “

” is attached, specifically both the bonds “

” and “

” are encompassed by the meaning of “

”.

Any formula given herein is also intended to represent unlabeled formsas well as isotopically labeled forms of the compounds. Isotopicallylabeled compounds have structures depicted by the formulas given hereinexcept that one or more atoms are replaced by an atom having a selectedatomic mass or mass number. Examples of isotopes that can beincorporated into compounds of the disclosure include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, andiodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S,¹⁸F, ³⁶Cl, and ¹²⁵I, respectively. Such isotopically labelled compoundsare useful in metabolic studies (preferably with ¹⁴C), reaction kineticstudies (with, for example ²H or ³H), detection or imaging techniques[such as positron emission tomography (PET) or single-photon emissioncomputed tomography (SPECT)] including drug or substrate tissuedistribution assays, or in radioactive treatment of patients. Further,substitution with heavier isotopes such as deuterium (i.e., ²H) mayafford certain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements. Isotopically labeled compounds of this disclosure andprodrugs thereof can generally be prepared by carrying out theprocedures disclosed in the schemes or in the examples and preparationsdescribed below by substituting a readily available isotopically labeledreagent for a non-isotopically labeled reagent.

Any disubstituent referred to herein is meant to encompass the variousattachment possibilities when more than one of such possibilities areallowed. For example, reference to disubstituent -A-B-, where A≠B,refers herein to such disubstituent with A attached to a firstsubstituted member and B attached to a second substituted member, and italso refers to such disubstituent with A attached to the secondsubstituted member and B attached to the first substituted member.

MATERIALS AND METHODS

Synthesis of MBQ-167

All reagents were purchased from Sigma-Aldrich Chemical Company. Thesynthesis of 3-Azido-9-ethyl-9H-carbazole 3 is as follows (FIG. 1).

Step 1: To a solution of 2.10 g (10.0 mmol)9-Ethyl-9H-carbazol-3-yl-amine 1 in 20 mL water, 2.0 mL (40.0 mmol) ofconcentrated sulfuric acid (H₂SO₄) was added. When all the amine wasconverted to the sulfate (green precipitate), 10 mL more of water wasadded and the suspension cooled to 0-5° C. in an ice-water bath. Asolution of 0.828 g (12.0 mmol) sodium nitrite (NaNO₂) in 5 mL of waterwas added dropwise, and the mixture stirred for 1 h. Next, a solution of0.780 g (12.0 mmol) of sodium azide (NaN₃) in 5 mL of water was addeddropwise and stirred continuously for 2-8 h. After completion of thereaction, the reaction mixture was warmed to 25° C., 30 mL of ethylacetate and 20 mL of distilled water were added, and after vigorousmixing, the layers were separated. The organic layer was extracted with10 mL brine, separated, dried on sodium sulfate, filtered andconcentrated on a rotary evaporator. After silica gel chromatographyusing 3:1 hexanes/ethyl acetate as the eluent,3-Azido-9-ethyl-9H-carbazole 3 was obtained as an off-white solid in ayield of 1.79 g (7.58 mmol=76%). The product was identified with TLC andNMR spectroscopy. R_(f)=0.82 (3:1, Hexane/Ethyl Acetate); ¹HNMR (CDCl₃,400 MHz) δ 1.43 (t, J=7.19 Hz, 3H), 4.37 (q, J=7.19 Hz, 2H), 7.14 (dd,J=2.17, 8.63 Hz, 1H), 7.24 (d, J=7.13 Hz, 1H), 7.37 (d, J=8.64 Hz, 1H),7.41 (d, J=8.24 Hz, 1H), 7.49 (t, J=7.14 Hz, 1H), 7.75 (d, J=2.19 Hz,1H), 8.07 (d, J=7.80 Hz, 1H); ¹³C (CDCl₃, 100 MHz) δ 14.1, 38.0, 109.0,109.8, 110.7, 117.5, 119.3, 120.9, 122.5, 124.2, 126.6, 131.4, 137.8140.9.

Step 2: Synthesis of1-(9-Ethyl-9H-carbazol-3-yl)-5-phenyl-1H-1,2,3-triazole 6 (MBQ-167). Ina 25 mL 3-neck round bottom flask containing phenylacetylene 0.11 g (1.1mmol) under a nitrogen atmosphere, a solution of ethylmagnesium bromidein THF 1.1 mL (1.1 mmol) was added dropwise at 25° C. After the Grignardreagent was added, the mixture was heated at 50° C. for 15 min andcooled to 25° C. A solution of 0.24 g (1.0 mmol) of azide 3 in THF (1.0M) was added dropwise and heated to 50° C. for 1 h. After quenching with10% ammonium chloride, the products were extracted with ethyl acetate(3×). The organic layer was washed with 10 mL of brine, separated anddried on sodium sulfate, filtered and concentrated on a rotaryevaporator to obtain crude material (0.33 g). The crude oil was purifiedvia silica gel chromatography to obtain 0.29 g (0.86 mmol=86%) of1-(9-Ethyl-9H-carbazol-3-yl)-5-phenyl-1H-1,2,3-triazole MBQ-167 as awhite solid. Purity (≥98%) was verified by TLC, NMR spectroscopy, andGC/MS: R_(f)=0.26 (3:1, hexane/ethyl acetate); ¹HNMR (CDCl₃, 400 MHz) δ1.47 (t, J=7.22 Hz, 3H), 4.38 (q, J=7.22 Hz, 2H), 7.26-7.33 (m, 6H),7.36 (dt, J=1.76, 8.60 Hz, 1H), 7.41 (d, J=8.84 Hz, 1H), 7.46 (d, J=8.32Hz, 1H), 7.53 (t, J=7.32 Hz, 1H), 7.93 (s, 1H), 8.20 (d, J=7.84 Hz, 1H),8.15 (d, J=1.80 Hz, 1H); ¹³C (CDCl₃, 100 MHz) δ 13.8, 37.9, 108.7,108.9, 117.9, 119.5, 120.8, 122.5, 123.0, 123.2, 126.6, 127.1, 128.3,128.5, 128.8, 129.0, 133.1, 138.0, 139.8, 140.7. LRGC-MS m/z (rel %):[M]⁺ 338 (37), [M-C₂H₅]⁺ 310 (55), [M-C₂H₅N]⁺ 295 (100), [M-C₂H₅N₂]⁺ 281(34), [M-C₉H₉N₃]⁺ 179 (34).

9-Ethyl-3-(5-phenyl-1H-1,2,3-triazol-1-yl)-9H-carbazole (I-1).

Step 1: Synthesis of 3-Azido-9-ethyl-9H-carbazole: To a slurry of 2.10 g(10.0 mmol) 9-Ethyl-9H-carbazol-3-yl-amine in 20 mL water, 2.0 mL (40.0mmol) of concentrated sulfuric acid (H₂SO₄) was added. When all theamine has been converted to the sulfate (green precipitate), 10 mL moreof water was added and the suspension is cooled to 0-5° C. in anice-water bath. A solution of 0.828 g (12.0 mmol) of sodium nitrite(NaNO₂) in 5 mL of water was added dropwise, and the mixture is stirredfor 1 h. With strong stirring, a solution of 0.780 g (12.0 mmol) ofsodium azide (NaN₃) in 5 mL of water was added dropwise and stirring iscontinued for 2-8 h. After the reaction mixture was warmed to roomtemperature, 30 mL of ethyl acetate and 20 mL of distilled water wereadded, and after vigorous mixing, the layers were separated with the aidof a separation funnel. The organic layer was extracted with 10 mLbrine, separated and dried on sodium sulfate, filtered and concentratedon a rotary evaporator to obtain a crude brown oil. After silica gelchromatography using 3:1 hexanes/ethyl acetate as the eluent, the3-Azido-9-ethyl-9H-carbazole 2 was obtained as a pure compound in ayield of 1.79 g (7.58 mmol=76% from crude material). The product wasidentified with TLC, NMR and GC/MS. R_(f)=1.00 (3:1, Hexane/EthylAcetate); ¹H NMR (CDCl₃, 400 MHz) δ 1.43 (t, J=7.26 Hz, 3H), 4.37 (q,J=7.24 Hz, 2H), 7.14 (dd, J=2.17, 8.63 Hz, 1H), 7.24 (t, J=7.13 Hz, 1H),7.37 (d, J=8.64 Hz, 1H), 7.41 (d, J=8.24 Hz, 1H), 7.49 (t, J=8.17 Hz,1H), 7.75 (d, J=2.19 Hz, 1H), 8.07 (d, J=7.80 Hz, 1H); ¹³C (CDCl₃, 100MHz) δ 13.7, 37.6, 109.2, 108.6, 109.4, 110.3, 117.1, 118.9, 120.2,120.6, 122.1, 123.8, 126.2, 131.0, 137.5, 140.5.

Step 2: Synthesis of1-(9-Ethyl-9H-carbazol-3-yl)-5-phenyl-1H-1,2,3-triazole (I-1). To a 25mL 3-neck round bottom flask containing phenylacetylene 0.11 g (1.1mmol) under a nitrogen atmosphere, a solution of ethylmagnesium bromide(EtMgBr) in THF 1.1 mL (1.1 mmol) was added dropwise at roomtemperature. After the ethylmagnesium bromide was added, the mixture washeated at 50° C. for 15 min and cooled to room temperature. A solutionof 0.24 g (1.0 mmol) of azide 2 in THF (1.0 M) was added dropwise andthe mixture heated to 50° C. for 1 h. After quenching with a solution of10% ammonium chloride (NH₄Cl), the products were extracted with ethylacetate (3×). The organic layer was washed with 10 mL of brine,separated and dried on sodium sulfate, filtered and concentrated on arotary evaporator to obtain crude material (0.1104 g). The crude oil waspurified via silica gel chromatography to obtain 0.29 g (0.86 mmol=86%)of 1-(9-Ethyl-9H-carbazol-3-yl)-5-phenyl-1H-1,2,3-triazole (I-1). Theproduct was identified to be essentially pure by TLC and NMR: R_(f)=0.26(3:1, hexane/ethyl acetate); ¹H NMR (CDCl₃, 400 MHz) δ 1.45 (t, J=6.64Hz, 3H), 4.38 (q, J=7.24 Hz, 2H), 7.25 (d, J=5.5, 1H), 7.27-7.29 (m,4H), 7.31 (t, J=1.7 Hz, 1H), 7.36 (dd, J=1.9, 8.6 Hz, 1H), 7.39 (d,J=8.6 Hz, 1H), 7.44 (d, J=8.2 Hz, 1H), 7.52 (t, J=7.2 Hz, 1H); ¹³C(CDCl₃, 100 MHz) δ 13.8, 37.8, 53.4, 108.7, 108.9, 117.8, 119.5, 120.7,122.0, 122.9, 123.1, 126.6, 127.0, 128.3, 128.5, 128.7, 128.9, 133.0,138.0, 139.7, 140.6. LRGC-MS m/z (rel %): [M]⁺ 338 (37), [M-C2H5]⁺ 310(55), [M-C2H5N]⁺ 295 (100), [M-C2H5N2]⁺ ²⁸¹ (34), [M-C9H9N3]⁺ 179 (34).

Step 2: Synthesis of1-(9-Ethyl-9H-carbazol-3-yl)-5-phenyl-1H-1,2,3-triazole (I-1). To a 25mL 3-neck round bottom flask containing phenylacetylene 0.11 g (1.1mmol) under a nitrogen atmosphere, a solution of ethylmagnesium bromide(EtMgBr) in THF 1.1 mL (1.1 mmol) was added dropwise at roomtemperature. After the ethylmagnesium bromide was added, the mixture washeated at 50° C. for 15 min and cooled to room temperature. A solutionof 0.24 g (1.0 mmol) of azide 2 in THF (1.0 M) was added dropwise andthe mixture heated to 50° C. for 1 h. After quenching with a solution of10% ammonium chloride (NH4Cl), the products were extracted with ethylacetate (3×). The organic layer was washed with 10 mL of brine,separated and dried on sodium sulfate, filtered and concentrated on arotary evaporator to obtain crude material (0.1104 g). The crude oil waspurified via silica gel chromatography to obtain 0.29 g (0.86 mmol=86%)of 1-(9-Ethyl-9H-carbazol-3-yl)-5-phenyl-1H-1,2,3-triazole (I-1). Theproduct was identified to be essentially pure by TLC and NMR: R_(f)=0.26(3:1, hexane/ethyl acetate); ¹H NMR (CDCl₃, 400 MHz) δ 1.45 (t, J=6.64Hz, 3H), 4.38 (q, J=7.24 Hz, 2H), 7.25 (d, J=5.5, 1H), 7.27-7.29 (m,4H), 7.31 (t, J=1.7 Hz, 1H), 7.36 (dd, J=1.9, 8.6 Hz, 1H), 7.39 (d,J=8.6 Hz, 1H), 7.44 (d, J=8.2 Hz, 1H), 7.52 (t, J=7.2 Hz, 1H); ¹³C(CDCl₃, 100 MHz) δ 13.8, 37.8, 53.4, 108.7, 108.9, 117.8, 119.5, 120.7,122.0, 122.9, 123.1, 126.6, 127.0, 128.3, 128.5, 128.7, 128.9, 133.0,138.0, 139.7, 140.6. LRGC-MS m/z (rel %): [M]⁺ 338 (37), [M-C2H5]⁺ 310(55), [M-C2H5N]⁺ 295 (100), [M-C2H5N2]⁺ 281 (34), [M-C9H9N3]⁺ 179 (34).

Cell Culture

MDA-MB-231, MCF-7 (ATCC), green fluorescent protein (GFP) tagged bonemetastatic variant of MDA-MB-435 (GFP-HER2-BM) (characterized in (25),from Dr. Danny Welch, The University of Kansas Cancer Center), andMCF10A mammary epithelial cells (ATCC) were cultured and maintained aspreviously described (16). MDA-MB-231 and MCF-7 cell lines were obtainedin 2000, the MCF-10A cell line was purchased in 2013, and theGFP-HER2-BM cell line was a gift from Dr. Danny Welch in 2008. The celllines were authenticated by ATCC in 2015.

Rac and Cdc42 Activation Assays

For the IC₅₀ curves: Rac1/2/3 and Cdc42 activation was determined asdescribed (16), using a G-LISA kit (Cytoskeleton, Inc., Denver, Colo.).MDA-MB-231 cell lysates were prepared from 24 h MBQ-167 treatment bycombining attached and detached cell populations (N=3). Four-parameterdose-response IC₅₀ curves were fitted using the nonlinear regressionfunction of GraphPad Prism®.

Additionally, Rac, Cdc42, or Rac activation was determined, by pulldownsusing the P21-binding domain (PBD) of PAK, or Rho binding domain ofRhotekin as described (2, 16). The GTP bound active Rac, Cdc42, or Rhowas detected by Western blot (N=3).

Western Blot Analysis

Total cell lysates or pull-downs were Western blotted using routineprocedures. The primary antibodies used were: Rac (Rac1,2,3), Cdc42,Bcl-xL, Bcl-2, Mcl-1, PAK1, PAK2, phospho (p)-PAK1(T423)/PAK2(T402),p-PAK1(S199/204)/PAK2(S192/197), p-PAK1(S144/204)/PAK2(S141), LIM kinase(LIMK1), p-LIMK1/2(Tyr507/Thr508), Cofilin, p-cofilin(S3), STAT3,p-STAT3(Y705), p-P-38 MAPK (T180/Y182), p-ERK (T202/Y204), p-Akt (S473),and Akt (Cell Signaling Technology, Inc.) and β-actin (Sigma).

Fluorescence Microscopy

MDA-MB-231 cells were treated with vehicle or MBQ-167 at 250 or 500 nMfor 24 h. Cells were fixed, permeabilized, and stained with Rhodaminephalloidin to visualize F-actin, and with p-tyrosine or vinculin tovisualize focal adhesions, as described (2). Fluorescence micrographswere acquired at 600× in an Olympus BX40 fluorescence microscope using aSpot digital camera.

Cell Migration Assays

Transwell Assay:

As described (2), quiescent MDA-MB-231 cells were treated with vehicleor MBQ-167 (250 nM) for 24 h. The attached and detached populations wereseparated and exactly 2×10⁵ cells were placed on the top well ofTranswell chambers with 5% FBS in the bottom well. The number of cellsthat migrated to the underside of the membrane following a 7 hincubation was quantified after staining fixed cells with propidiumiodide (PI). For each treatment (N=3), cells in 20 microscopic fieldswere quantified.

Wound Healing Scratch Assay:

MDA-MB-231 cells plated on 6-well plates at equal cell density wereincubated in 10% FBS until confluent. The media was changed to 2% FBSand a single scratch was made in the center of the monolayer culturewith a pipet tip. MBQ-167 was added at 0, 250, or 500 nM immediatelyfollowing wounding. Images were digitally acquired from an Olympusmicroscope (4× magnification) at 0, 8, 12, and 24 h and the scratchdistance quantified in Adobe Photoshop. N=3 biological replicates (with2 technical replicates each).

Mammosphere Formation Assay

As described (26), Equal numbers of MDA-MB-231 cells treated withvehicle or MBQ-167 were seeded in ultra-low attachment plates (Corning)at a density of 500 cells/well in serum-free mammary epithelium basalmedium (Lonza). Mammospheres were counted after 4 days incubation in 0or 250 nM MBQ-167 at 37° C., 5% CO₂. Mammosphere-forming efficiency wascalculated as the number of mammospheres divided by the number of cellsseeded per well and expressed relative to vehicle controls.

Cell Viability Assays

As described (16), equal numbers of MDA-MB-231, GFP-HER2-BM, or MCF-10Acells were incubated in 0-1 μM MBQ-167 for 120 h. The CellTiter 96®Non-Radioactive Cell Proliferation Assay (Promega, Fitchburg, Wis.) wasused according to the manufacturer's instructions. This assay allows thequantification of the viability of both attached and detached cells inthe same well. GI₅₀ was determined as 100×(T−T₀)/(C−T₀)=50 (T=theoptical density of drug treatment after 120 h, T₀=the optical density attime zero, and C=the optical density of the untreated cells). Curveswere fitted using the four-parameter logistic nonlinear regressionmodels in GraphPad Prism software.

Cell Cycle Progression

MDA-MB-231 cells were incubated with 0 or 250 nM MBQ-167 for 48 h andall cells (detached and attached) were stained with PI, as in (27). Cellcycle stage was analyzed using a four-color flow cytometer (FACSCalibur,BD Biosciences, San Jose, Calif.). A total of 20,000 events wereanalyzed for each sample. List-mode files were collected using CellQuest software 3.3 and analyzed using the Flow Jo software vX.0.7 (BDBiosciences, San Jose, Calif.).

Apoptosis Assay

Apoptosis was measured using a Caspase-Glo3/7 Luminescence Assay Kit asper manufacturer's instructions (Promega, Corp., Madison, Wis., USA).Following treatment of equal numbers of cells with vehicle or MBQ-167for 24 h, Caspase-3/7 Glo reagent was added and incubated at roomtemperature for 60 min. Caspase-3/7 activities were determined byquantifying luminescence.

Annexin V Staining

Apoptotic cells were detected by fluorescence microscopy of AnnexinV-Cy3-18 stained cells as per manufacturer's instructions(Sigma-Aldrich, St Louis, Mo., USA). Briefly, GFP-MDA-MB-231 cells grownon coverslips were treated with vehicle, or 250 or 500 nM MBQ-167 for 6h and stained with Annexin V-Cy3-18 in binding buffer (10 mM HEPES/NaOH,pH 7.5, 0.14 M NaCl, 2.5 mM CaCl₂) for 15 min at room temperature.Coverslips were washed in binding buffer and fixed with 3.7%paraformaldehyde prior to fluorescence microscopy. Images were digitallyacquired from an Olympus inverted fluorescence microscope.

Animal Protocol

All animal studies were conducted under approved protocol #A8180112Institutional Animal Care and Use Committee, in accordance with the NIHGuideline for the Care and Use of Laboratory Animals. Female athymicnu/nu mice and severe combined immunodeficiency Crl:SHO-Prkdc SCIDHairless 4 to 5 wk old (Charles River Laboratories, Inc., Wilmington,Mass.) were maintained under pathogen-free conditions in HEPA-filteredcages.

Tumor Establishment

GFP-HER2-BM cells (˜5×10⁵) or GFP-MDA-MB-231 cells (1×10⁵) in Matrigel(BD Biosciences, San Jose, Calif.) were injected at the fourth rightmammary fat pad under isofluorane inhalation (1-3% in oxygen using aninhalation chamber at 2 L/min) to produce orthotopic primary tumors.After tumor establishment (1 wk post-inoculation), animals were randomlydivided into treatment groups (n=6).

Administration of MBQ-167

Mice were treated with vehicle (12.5% ethanol, 12.5% Cremophor(Sigma-Aldrich, St. Louis, Mo.), and 75% 1×PBS pH 7.4), or 1 or 10 mg/kgBW MBQ-167 by i.p. injection in a 100 μL volume 3× a wk. Treatmentscontinued until sacrifice at day 65.

Whole Body Fluorescence Image Analysis

Mammary tumor growth was quantified as changes in the integrated densityof GFP fluorescence, as in (28). Mice were imaged on day 1 of treatmentadministration, and once a week thereafter for 65 days, using theFluorVivo small animal in vivo imaging system (INDEC Systems, Inc.,Santa Clara, Calif.). Tumor fluorescence intensities were analyzed usingImage J software (National Institutes of Health, Bethesda, Md.).Relative tumor growth was calculated as the integrated density offluorescence of each tumor on each day of imaging relative to theintegrated density of fluorescence of the same tumor on day 1 oftreatment, as described (17). As in (29), Optimal Tumor growth wascalculated as % T/C=(δT/δC)×100 when δT>0, δT=average tumor size on day65 of treated mice−average tumor size on day 01 of treated mice.δC=average tumor size on day 65 of control mice−average tumor size onday 01 of control mice. Tumor growth delay was calculated as thepercentage by which the treated group tumor size is delayed in attaininga specified number of doublings (from day 1) compared with controlsusing: [(T−C)/C]×100, where T and C are the median times in days fortreated and control groups to double in tumor size.

Analysis of Metastases

Following sacrifice, lungs, kidneys, livers, bones, and spleens wereexcised and immediately stored in liquid N₂. Stored organs were thawedand analyzed by fluorescence microscopy, as described.

Liver Enzyme Assays

Frozen stored livers were thawed and homogenized to measure alkalinephosphatase (ALP) and alanine transaminase (ALT) activities usingcolorimetric assay kits from Abcam and Cayman Chemicals respectively, asper manufacturer's instructions.

Statistical Analysis

Statistical analyses used Microsoft Excel and GraphPad Prism, anddifferences were considered statistically significant at P≤0.05.

TABLE 1A TABLE I Cmpd Rac Cdc42 # Structure activity activity ¹H-NMRName I-1 

A A (CDCl₃) δ 1.43 (t, J = 7.26 Hz, 3H), 4.37 (q, J = 7.24 Hz, 2H), 7.14(dd, J = 2.17, 8.63 Hz, 1H), 7.24 (t, J = 7.13 Hz, 1H), 7.37 (d, J =8.64 Hz, 1H), 7.41 (d, J = 8.24 Hz, 1H), 7.49 (t, J = 8.17 Hz, 1H), 7.75(d, J = 2.19 Hz, 1H), 8.07 (d, J = 7.80 Hz, 1H) 9-ethyl-3-(5-phenyl)-1H- 1,2,3-triazol-1- yl)-9H-carbazole I-2 

(CDCl₃) δ 1.45 (t, J = 7.24 Hz, 3H), 4.38 (q, J = 7.24 Hz, 2H), 6.99(dd, J = 1.4, 8.76, 1H), 7.12-7.17 (m, 3H), 7.24 (t, J = 7.0 Hz, 1H),7.34 (dd, J = 2.0, 8.6 Hz, 1H), 7.39 (d, J = 8.6 Hz, 1H), 7.45 (d, J =8.2 Hz, 1H), 7.51 (t, J = 7.2 Hz, 1H), 7.91 (s, 1H), 8.01 (d, J = 7.8Hz, 1H), 8.17 (d, J = 1.9 Hz, 1H) 9-ethyl-3-(5-(m- tolyl)-1H-1,2,3-triazol-1-yl)-9H- carbazole I-3 

(CDCl₃) δ 1.45 (t, J = 7.20 Hz, 3H), 3.63 (s, 3H), 4.39 (q, J = 7.20 Hz,2H), 6.81 (dd, J = 1.4, 4.5, 1H), 6.85 (dd, J = 2.5, 9.5 Hz, 1H), 7.19(t, J = 8.0 Hz, 1H), 7.36 (dd, J = 2.0, 8.6 Hz, 1H), 7.41 (d, J = 8.6Hz, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.52 (t, J = 7.2 Hz, 1H), 7.92, (s,1H), 8.20 (d, J = 7.8 Hz, 1H), 8.16 (d, J = 1.9 Hz, 1H);9-ethyl-3-(5-(3- methoxyphenyl)- 1H-1,2,3-triazol- 1-yl)-9H- carbazoleI-4 

I-5 

I-6 

I-7 

I-8 

I-9 

I-10

I-11

I-12

I-13

I-14

I-15

I-16

I-17

I-18

I-19

I-20

I-21

I-22

I-23

I-24

I-25

I-26

I-27

I-28

I-29

I-30

I-31

I-32

I-33

I-34

I-35

I-36

I-37

I-38

I-39

I-40

I-41

I-42

I-43

I-44 I-45 I-46 I-47 I-48 I-49 I-50 I-51 A: >50% inhibition at 250 nM;B: >50% inhibition at 1 μM; C >50% inhibition at 5 μM; D <50% inhibitionat 5 μM.

TABLE 1B Migration GI₅₀ (μM) Inhibition (%) Cmpd MDA- MDA-MB- #Structure MB-231 231 ¹H-NMR and ¹³C-NMR Name I-1 

0.128 37.2 (0.25 μM) ¹H-NMR (400 MHz, CDCl₃) δ 1.43 (t, J = 7.3, 3H),4.37 (q, J = 7.2, 2H), 7.14 (dd, J = 2.17, 8.63, 1H), 7.24 (t, J = 7.13,1H), 7.37 (d, J = 8.64, 1H), 7.41 (d, J = 8.24, 1H), 7.49 (t, J = 8.17,1H), 7.75 (d, J = 2.19, 1H), 8.07 (d, J = 7.80, 1H); ¹³C NMR (100 MHz,CDCl₃) δ 13.8, 37.8, 108.7, 108.9, 117.8, 119.5, 120.8, 122.5, 123.0,123.2, 126.6, 127.1, 128.3, 128.5, 128.8, 128.0, 133.0, 138.0, 139.8,140.7. 9-ethyl-3-(5- phenyl-1H-1,2,3- triazol-1-yl)-9H- carbazole I-2 

1.332 ≤1     (1 μM) ¹H-NMR (400 MHz, CDCl₃) δ 1.45 (t, J = 7.24, 3H),4.38 (q, J = 7.24, 2H), 6.99 (dd, J = 1.4, 8.76, 1H), 7.12-7.17 (m, 3H),7.24 (t, J = 7.0, 1H), 7.34 (dd, J = 2.0, 8.6, 1H), 7.39 (d, J = 8.6,1H), 7.45 (d, J = 8.2, 1H), 7.51 (t, J = 7.2, 1H), 7.91 (s, 1H), 8.01(d, J = 7.8, 1H), 8.17 (d, J = 1.9, 1H); ¹³C NMR (100 MHz, CDCl₃) δ14.0, 21.6, 38.1, 108.9, 109.2, 118.1, 119.8, 121.1, 122.8, 123.3,123.4, 125.9, 126.9, 127.2, 128.7, 128.9, 129.5, 130.0, 133.3, 138.4,138.8, 140.0, 140.9. 9-ethyl-3-(5-(m- tolyl)-1H-1,2,3- triazol-1-yl)-9H-carbazole I-3 

0.248 ≤1     (1 μM) ¹H-NMR (400 MHz, CDCl₃) δ 1.45 (t, J = 7.2, 3H),3.63 (s, 3H), 4.39 (q, J = 7.2, 2H), 6.81 (dd, J = 1.4, 4.5, 1H), 6.85(dd, J = 2.5, 9.5, 1H), 7.19 (t, J = 8.0, 1H), 7.36 (dd, J = 2.0, 8.6,1H), 7.41 (d, J = 8.6, 1H), 7.45 (d, J = 8.2, 1H), 7.52 (t, J = 7.2,1H), 7.92 (s, 1H), 8.20 (d, J = 7.8, 1H), 8.16 (d, J = 1.9, 1H); ¹³C NMR(100 MHz, CDCl₃) δ 13.8, 37.9, 55.4, 109.0, 109.1, 110.9, 113.3, 114.4,118.3, 118.6, 119.1, 119.6, 120.8, 122.6, 123.4, 126.7, 129.5, 129.9,131.9, 139.6, 140.9, 148.1, 160.1. 9-ethyl-3-(5-(3- methoxyphenyl)-1H-1,2,3-triazol-1- yl)-9H-carbazole I-4 

>50 65.1   (10 μM) ¹H-NMR (400 MHz, CDCl₃) δ 1.47 (t, 3H, J = 7.2), 4.39(q, J = 7.14, 2H), 7.2158 (d, 1H, J = 3.0), 7.26 (t, 1H, J = 7.4), 7.34(d, J = 1.8, 1H), 7.36 (d, 1H, J = 1.8), 7.42 (s, 1H), 7.45 (d, 1H, J =4.4), 7.52 (t, 1H, J = 7.3), 8.01 (d, 1H, J = 6.0), 8.12 (s, 1H), 8.56(d, J = 4.6, 1H), 8.62 (s, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 13.7, 37.8,108.9, 117.8, 119.6, 120.7, 122.2, 122.4, 122.2, 123.2, 123.4, 123.4,126.7, 149.9. 9-ethyl-3-(5- (pyridin-3-yl)-1H- 1,2,3-triazol-1-yl)-9H-carbazole I-5 

0.408  2.4   (1 μM) ¹H-NMR (400 MHz, CDCl₃) δ 1.50 (t, 3H, J = 7.2),4.43 (q, 2H, J = 7.239), 6.97 (d, J = 5.0, 1H), 7.09 (d, J = 2.84, 1H,),7.2720 (t, J = 3.0, 1H), 7.45 (d, J = 1.9, 1H), 7.48 (d, 1H, J = 6.137),7.53 (d, 1H, J = 7.2), 7.95 (s, 1H), 8.04 (d, 1H, J = 7.8), 8.16 (s,1H); ¹³C NMR (100 MHz, CDCl₃) δ 13.8, 37.9, 108.9, 108.9, 118.3, 119.7,120.8, 122.5, 123.2, 123.3, 124.3, 126.5, 126.7, 126.9, 127.0, 128.2,132.3, 134.1, 140.0, 140.7. 9-ethyl-3-(5- (thiophen-3-yl)-1H-1,2,3-triazol-1-yl)- 9H-carbazole I-6 

>50 28     (10 μM) ¹H-NMR (400 MHz, CDCl₃) δ 1.48 (t, 3H, J = 7.1), 4.43(q, 2H, J = 7.3), 7.29 (t, 1H, J = 13.5), 7.47 (d, 1H, J = 8.1), 7.53(t, 1H, J = 7.8), 7.83 (t, 1H, J = 7.8), 7.87 (d, 1H, J = 2.11), 7.89(d, 1H, J = 2.1), 8.13 (d, 1H, J = 7.36), 8.29 (d, 1H, J = 7.2), 8.49(s, 1H), 8.64 (d, 1H, J = 4.1), 8.68 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ13.8, 37.9, 108.9, 109.1, 113.2, 118.8, 119.6, 120.4, 120.7, 120.8,122.6, 122.9, 123.4, 126.7, 129.4, 136.9, 139.7, 140.8, 148.8, 149.5,150.3. 9-ethyl-3-(5- (pyridin-2-yl)-1H- 1,2,3-triazol-1-yl)-9H-carbazole I-7 

>50 30.8   (10 μM) ¹H-NMR (400 MHz, CDCl₃) δ 1.48 (t, 3H, J = 7.3), 4.41(q, 2H, J = 5.652), 6.8907 (d, 1H, J = 7.9), 7.9872 (d, 1H, J = 3.0),7.00 (d, 1H, J = 4.2), 7.09 (t, 1H, J = 8.3), 7.21 (d, 1H, J = 8.6),7.26 (t, 1H, J = 9.7), 7.32 (t, 1H, J = 7.7), 7.41 (d, 1H, J = 6.4),7.46 (d, 1H, J = 9.7), 7.53 (t, 1H, J = 6.5), 7.90 (s, 1H), 8.04 (d, 1H,J = 4.3), 8.13 (s, 1H); ¹³C NMR (100 MHz, CDCl3) δ 13.9, 37.9, 108.8,108.9, 117.3, 117.918, 118.405, 119.604, 120.239, 120.8, 121.5, 122.5,123.1, 123.2, 124.1, 124.7, 126.7, 128.3, 129.9, 130.0, 130.1, 132.4,132.8, 138.1, 139.8, 140.7. 9-ethyl-3-[5-(4- phenoxy-phenyl)-1H-1,2,3-triazol-1- yl)]-9H-carbazole I-8 

>50 n/a ¹H-NMR (400 MHz, CDCl₃) δ 1.46 (t, J = 7.6, 3H), 2.96 (t, J =6.3, 2H), 3.85 (t, J = 6.3, 2H), 4.41 (q, J = 7.6, 2H), 7.25 (dd, J =7.6, 1H), 7.44-7.55 (m, 4H), 7.71 (s, 1H), 8.04 (d, J = 7.6, 1H), 8.14(t, J = 1.4, 1H). 2-(1-(9-ethyl-9H- carbazol-3-yl)-1H- 1,2,3-triazol-5-yl)ethan-1-ol I-9 

n/a n/a ¹H-NMR (400 MHz, CDCl₃) δ 1.49 (t, J = 7.2, 3H), 2.95 (s, 3H),3.20 (t, J = 6.4, 2H), 4.38 (t, J = 6.4, 2H), 4.45 (q, J = 7.2, 2H),7.30 (dd, J = 7.2, 1H), 7.43-7.58 (m, 4H), 7.77 (s, 1H), 8.10 (d, J =8.0, 1H), 8.13 (t, J = 1.6, 1H) 2-(1-(9-ethyl-9H- carbazol-3-yl)-1H-1,2,3-triazol-5- yl)ethyl methanesulfonate I-10

n/a n/a ¹H-NMR (400 MHz, CDCl₃) δ 1.48 (t, J = 7.2, 3H), 2.95 (s, 3H),3.20 (t, J = 6.4, 2H), 4.43 (t, J = 6.4, 2H), 4.45 (q, J = 7.2, 2H),5.47 (d, J = 11.4, 1H), 5.84 (d, J = 17.6, 1H), 6.52 (dd, J = 17.6 and11.4 H, 1H), 7.29 (dd, J = 7.2, 1H), 7.47-7.57 (m, 4H), 7.95 (s, 1H),8.10 (d, J = 7.8, 1H), 8.18 (t, J = 1.6, 1H) 9-ethyl-3-(5-vinyl-1H-1,2,3-triazol-1- yl)-9H-carbazole I-11

n/a n/a ¹H-NMR (400 MHz, CDCl₃) δ 1.40 (t, J = 7.2, 3H), 1.80 (q, J =7.6, 2H), 2.75 (t, J = 7.8, 2H), 3.78 (t, J = 6.2, 2H), 4.32 (q, J =7.2, 2H), 7.23 (dd, J = 7.6, 1H), 7.44-7.55 (m, 4H), 7.60 (s, 1H), 8.03(d, J = 7.6, 1H), 8.13 (s, 1H) 4-(2-(1-(9-ethyl- 9H-carbazol-3-yl)-1H-1,2,3-triazol-5- yl)ethyl)morpholine I-12

n/a n/a ¹H-NMR (400 MHz, CDCl₃) δ 1.49 (t, J = 7.6, 3H), 2.39 (m, 4H),2.60 (t, J = 7.6, 2H), 2,88 (t, J = 7.6, 2H), 3.66 (m, 4H), 4.45 (q, J =7.6, 2H), 7.29 (dd, J = 7.6, 1H), 7.47-7.58 (m, 4H), 7.71 (s, 1H), 8.11(d, J = 7.6, 1H), 8.15 (d, J = 1.4, 1H). 4-(2-(1-(9-ethyl-9H-carbazol-3-yl)- 1H-1,2,3-triazol-5- yl)ethyl)morpholine

TABLE 2 Effect of MBQ-167 on Rho GTPase activity (Active RhoGTPase/Total Rho GTPase) following 24 h in MDA-MB-231 cells, relative tovehicle (=1) Rho GTPase Attached cells Detached cells Rac 0.74* 0.23*Cdc42 0.39* 0.22* Rho 0.9 0.85 *p < 0.05

MDA-MB-231 human breast cancer cells were treated for 24 h with 250 nMMBQ-167. The attached and detached cell populations were recovered andequal amounts of proteins subjected to pulldown assays using thep21-binding domain of PAK to isolate the GTP bound Rac and Cdc42, or theRho binding domain from Rhotekin to isolate active Rho. Cell lysateswere western blotted with antibodies to Rac, Cdc42, or Rho. Results frompositive bands in western blots were quantified using image J. Theintegrated density for active Rho GTPase (Rac, Cdc42, Rho.GTP) wasdivided by the total Rho GTPase (Rac, Cdc42, Rho) from the same celllysates. Rac, Cdc42, or Rho activity for each MBQ-167 treatment wasdivided by the vehicle controls for each experiment to obtain RelativeRho GTPase activity.

TABLE 3 Effect of MBQ-167 on Rac activity (Active Rac/Total Rac)following 24 h in MCF-7 cells MBQ-167 nM Rac Activity 0 1.0 250 1.075 ±0.3

MCF-7 human breast cancer cells were treated for 24 h with 250 nMMBQ-167. Cells were lysed and equal amounts of proteins subjected topulldown assays using the p21-binding domain of PAK to isolate the GTPbound Rac. Cell lysates were western blotted with antibodies to Rac,Cdc42, or Rho. Results from positive bands in western blots werequantified using image J. The integrated density for active Rac(Rac.GTP) was divided by the total Rac from the same cell lysates (N=2).

TABLE 4 Optimal % Treated (T)/Control (C) for mammary fat pad tumorgrowth for MBQ-167 treated nude mice. Treatment mg/kg BW Optimal %Change in Tumor Size 0 100 1 58 10 9

As adapted from (Alley et al., Chapter 7, Human tumor xenograft modelsin NCI drug development, from Anticancer drug development guide B ATeicher and P A Andrews, Eds, 2004, Humana Press, Inc., NJ), % Changefor tumors from mice that received MBQ-167 treatment was calculated as:% T/C=(delta T/delta C)×100 when delta T>0

Delta T=average tumor size on day 65 of treated mice−average tumor sizeon day 01 of treated mice. Delta C=average tumor size on day 65 ofcontrol mice−average tumor size on day 01 of control mice.

TABLE 5 Tumor growth delay for MBQ-167-treated mice Time in Days forDoubling of Tumor size Treatment mg/kg BW 2¹ 2² 2³ 2⁴ 0 8 14.5 27.7 33.31 10 30 57 — 10 11 30 — —

As adapted from (Alley et al., Chapter 7, Human tumor xenograft modelsin NCI drug development, from Anticancer drug development guide B ATeicher and P A Andrews, Eds, 2004, Humana Press, Inc., NJ), Tumorgrowth delay was calculated as the percentage by which the treated grouptumor size is delayed in attaining a specified number of doublings (fromday 1) compared with controls using:

[(T−C)/C]×100, where T and C are the median times in days for treatedand control groups to attain a doubling in tumor size.

PUBLICATIONS

These publications are incorporated by reference to the extent theyrelate materials and methods disclosed herein.

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The invention claimed is:
 1. A compound of formula (II),

wherein R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆cycloalkyl, 3-to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, or mono- orbicyclic heteroaryl; wherein each hydrogen atom in C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to 7-memberedheterocycloalkyl, C₆-C₁₀ aryl, or mono- or bicyclic heteroaryl isindependently optionally substituted by deuterium, halogen, C₁-C₆ alkyl,C₁-C₆ haloalkyl, or —OR⁵; each R² is independently deuterium, halogen,—OH, —CN, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl,C₆-C₁₀ aryl, —OR⁶, —C(O)OR⁶, —C(O)NR⁶R⁷, —NH₂, —NH(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —CO₂H, —C(O)OC₁-C₆ alkyl,—C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl,—S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆ alkyl),—S(O)₂NH(C₁-C₆ alkyl); wherein each hydrogen atom in C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl and C₆-C₁₀ aryl isindependently optionally substituted by deuterium, halogen, —OH, —CN,—OR⁶, —OC₁-C₆ alkyl, —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂,—NHC(O)C₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂,—NHC(O)NHC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)NH₂, —N(C₁-C₆ alkyl)C(O)NHC₁-C₆alkyl, —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂,—NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)OC₁-C₆ alkyl, —NHS(O)(C₁-C₆alkyl), —NHS(O)₂(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl), —NHS(O)NH₂,—NHS(O)₂NH₂, —N(C₁-C₆alkyl)S(O)NH₂, —N(C₁-C₆ alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl),—NHS(O)₂NH(C₁-C₆ alkyl), —NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆alkyl)₂, —N(C₁-C₆ alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)S(O)₂NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)S(O)2N(C₁-C₆ alkyl)₂, —CO₂H, —C(O)OC₁-C₆ alkyl, —C(O)NH₂,—C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl),—S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆ alkyl)₂, —P(C₁-C₆ alkyl)₂,—P(O)(C₁-C₆ alkyl)₂, —C₃-C₆ cycloalkyl, or 3-to 7-memberedheterocycloalkyl; R³ is independently H, deuterium, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3-to 7-memberedheterocycloalkyl, C₆-C₁₀ aryl, C₆-C₁₀ aryl-(C₆-C₁₀ aryl), C₁-C₆alkyl-(NHR⁸), C₁-C₆ alkyl-(OR⁸), C₆-C₁₀ aryl-(OR⁸), C₁-C₆ alkyl-(NR⁸R⁹),or mono- or bicyclic heteroaryl; wherein each hydrogen atom in C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3-to 7-memberedheterocycloalkyl, C₆-C₁₀ aryl, or mono- or bicyclic heteroaryl isoptionally substituted by deuterium, halogen, —OH, oxo, —OR⁸, —NHR⁸,—CN, —OC₁-C₆ alkyl, —NH₂, —NH(C₁-C₆ alkyl), —NH(C₆-C₁₀ aryl), —NH(C₆-C₁₀aryl)-N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)C₁-C₆alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)NH₂,—N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl, —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆alkyl)C(O)OC₁-C₆ alkyl, —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),—NHS(O)NH₂,—NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆ alkyl)S(O)₂NH₂,—NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl), —NHS(O)N(C₁-C₆ alkyl)₂,—NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)S(O)₂NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)S(O)₂N(C₁-C₆ alkyl)₂, —CO₂H, —C(O)OC₁-C₆ alkyl, —C(O)NH₂,—C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl),—S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆ alkyl)₂, —P(C₁-C₆ alkyl)₂,—P(O)(C₁-C₆ alkyl)₂, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, —C₃-C₆cycloalkyl, 3-to 7-membered heterocycloalkyl, C₁-C₆ alkyl-(3-to7-membered heterocycloalkyl), —CF₃, —CHF₂, or —CH₂F; each R⁵, R⁶, R⁷,R⁸, and R⁹ is independently H, deuterium, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl,C₆-C₁₀ aryl, C₁-C₆ alkyl-(3-to 7-membered heterocycloalkyl), heteroaryl,—S(O)₂C₁-C₆ alkyl, —CF₃, —CHF₂, or —CH₂F, wherein each hydrogen inC₆-C₁₀ aryl is optionally substituted by C₃-C₆ cycloalkyl, 3- to7-membered heterocycloalkyl, or —CH₂CN; and x is 0, 1, 2, or 3; or apharmaceutically acceptable salt thereof.
 2. The compound of claim 1,wherein R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆cycloalkyl, or 3- to 7-membered heterocycloalkyl, wherein each hydrogenatom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, or3- to 7-membered heterocycloalkyl is independently optionallysubstituted by deuterium, halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or—OR⁵, or a pharmaceutically acceptable salt thereof.
 3. The compound ofclaim 2, wherein R¹ is H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, or 3-to7-membered heterocycloalkyl, wherein each hydrogen atom in C₁-C₆ alkyl,C₃-C₆ cycloalkyl, or 3- to 7-membered heterocycloalkyl is independentlyoptionally substituted by deuterium, halogen, C₁-C₆ alkyl, C₁-C₆haloalkyl, or —OR⁵, or a pharmaceutically acceptable salt thereof. 4.The compound of claim 3, wherein R¹ is H or C₁-C₆ alkyl, wherein eachhydrogen atom in C₁-C₆ alkyl is independently optionally substituted bydeuterium, halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or —OR⁵, or apharmaceutically acceptable salt thereof.
 5. The compound of claim 1,wherein R¹ is ethyl, or a pharmaceutically acceptable salt thereof. 6.The compound of claim 5, wherein x is 0, or a pharmaceuticallyacceptable salt thereof.
 7. The compound of claim 6, wherein R³ is C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3-to 7-memberedheterocycloalkyl, C₆-C₁₀ aryl, C₆-C₁₀ aryl-(C₆-C₁₀ aryl), C₁-C₆alkyl-(NHR⁸), C₁-C₆ alkyl-(OR⁸), C₆-C₁₀ aryl-(OR⁸), C₁-C₆ alkyl-(NR⁸R⁹),or mono- or bicyclic heteroaryl; wherein each hydrogen atom in C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3-to 7-memberedheterocycloalkyl, C₆-C₁₀ aryl, or mono- or bicyclic heteroaryl isoptionally substituted by deuterium, halogen, —OH, oxo, —OR⁸, —NHR⁸,—CN, —OC₁-C₆ alkyl, —NH₂, —NH(C₁-C₆ alkyl), —NH(C₆-C₁₀ aryl), —NH(C₆-C₁₀aryl)-N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)C₁-C₆alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)NH₂,—N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl, —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆alkyl)C(O)OC₁-C₆ alkyl, —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),—NHS(O)NH₂,—NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆ alkyl)S(O)₂NH₂,—NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl), —NHS(O)N(C₁-C₆ alkyl)₂,—NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)S(O)₂NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)S(O)₂N(C₁-C₆ alkyl)₂, —CO₂H, —C(O)OC₁-C₆ alkyl, —C(O)NH₂,—C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl),—S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆ alkyl)₂, —P(C₁-C₆ alkyl)₂,—P(O)(C₁-C₆ alkyl)₂, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, —C₃-C₆cycloalkyl, 3- to 7-membered heterocycloalkyl, C₁-C₆ alkyl-(3-to7-membered heterocycloalkyl), —CF₃, —CHF₂, or —CH₂F, or apharmaceutically acceptable salt thereof.
 8. The compound of claim 7,wherein R³ is C₆-C₁₀ aryl, C₆-C₁₀ aryl-(OR⁸), or mono- or bicyclicheteroaryl; wherein each hydrogen atom in C₆-C₁₀ aryl, or mono- orbicyclic heteroaryl is optionally substituted by deuterium, halogen,—OH, oxo, —OR⁸, —NHR⁸, —CN, —OC₁-C₆ alkyl, —NH₂, —NH(C₁-C₆ alkyl),—NH(C₆-C₁₀ aryl), —NH(C₆-C₁₀ aryl)-N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl,—N(C₁-C₆ alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl,—N(C₁-C₆ alkyl)C(O)NH₂, —N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl, —NHC(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)OC₁-C₆ alkyl,—N(C₁-C₆ alkyl)C(O)OC₁-C₆ alkyl, —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆alkyl), —N(C₁-C₆ alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆alkyl), —NHS(O)NH₂,—NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),—NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆ alkyl)₂, —CO₂H,—C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂,—SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆alkyl), —S(O)₂NH(C₁-C₆ alkyl), —S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆alkyl)₂, —P(C₁-C₆ alkyl)₂, —P(O)(C₁-C₆ alkyl)₂, C₁-C₆ alkyl, C₂- C₆alkenyl, C₂-C₆ alkynyl, —C₃-C₆ cycloalkyl, 3-to 7-memberedheterocycloalkyl, C₁-C₆ alkyl-(3- to 7-membered heterocycloalkyl), —CF₃,—CHF₂, or —CH₂F, or a pharmaceutically acceptable salt thereof.
 9. Thecompound of claim 8, wherein R³ is C₆-C₁₀ aryl, C₆-C₁₀ aryl-(OR⁸), ormonocyclic heteroaryl; wherein each hydrogen atom in C₆-C₁₀ aryl, ormono- or bicyclic heteroaryl is optionally substituted by deuterium,halogen, —OH, —OR⁸, —OC₁-C₆ alkyl, C₁-C₆ alkyl, or 3-to 7-memberedheterocycloalkyl, or a pharmaceutically acceptable salt thereof.
 10. Thecompound of claim 1, wherein the compound is selected from the groupconsisting of

or a pharmaceutically acceptable salt thereof.
 11. The compound of claim1, wherein the compound is selected from the group consisting of

or a pharmaceutically acceptable salt thereof.
 12. The compound of claim1, wherein the compound is of the formula

or a pharmaceutically acceptable salt thereof.
 13. The compound of claim1, wherein the compound is of the formula,

or a pharmaceutically acceptable salt thereof.
 14. The compound of claim1, wherein the compound is of the formula,

or a pharmaceutically acceptable salt thereof.
 15. The compound of claim1, wherein the compound is of the formula,

or a pharmaceutically acceptable salt thereof.
 16. A method of treatingcancer in a patient, the method comprising administering to the patientin need thereof an effective amount of a compound according to claim 1,wherein the cancer is selected from the group consisting of breastcancer, pancreatic cancer, ovarian cancer, gastric cancer, and neuronalcancer.
 17. The method of claim 16, wherein the cancer is gastriccancer.
 18. The method of claim 16, wherein the cancer is breast cancer.19. The method of of claim 16, wherein the compound is selected from thegroup consisting of

and, or a pharmaceutically acceptable salt thereof.
 20. A pharmaceuticalcomposition comprising a compound of formula

or a pharmaceutically acceptable salt thereof.